Viral vectors whose replication and, optionally, passenger gene are controlled by a gene switch activated by heat in the presence or absence of a small-molecule regulator

ABSTRACT

The present invention relates to conditionally replicating viruses or pairs of viruses containing a gene switch that is activatable by transient heat or other proteotoxic stress in the presence or absence of a small molecule regulator. The gene switch controls the expression of a gene for a protein required for efficient viral replication and may also control the activity of a passenger gene.

RELATED APPLICATIONS

This application is a division of application Ser. No. 11/148,368, filed Jun. 9, 2005, which is a continuation-in-part of application Ser. No. 10/731,961, filed Dec. 10, 2003.

TECHNICAL FIELD

The present invention relates to viral vectors whose replication and, optionally, passenger gene expression are regulated by a gene switch that is dually controlled by heat and a small-molecule regulator.

BACKGROUND OF THE INVENTION

There are numerous situations in biological research, ex vivo cell therapies and gene therapies of experimental animals and, eventually, humans, where careful control of the distribution and expression of an introduced gene or a replicating virus is critically important. Viral and non-viral vectors provide means for delivering genes to cells, tissues and organs. However, this delivery is, typically, not specific for any particular cell type, tissue or organ location. Previously, so-called tissue-specific promoters were used to restrict virus replication or expression of a gene delivered by a vector to cells of a specific type. A shortcoming of this approach is that is uncertain whether a “tissue-specific” promoter really exists. Although a promoter may have a much stronger activity in a particular cell type than in others, it is likely to display at least some activity in some other cells. Furthermore, the rate of transcription supported by a chosen tissue-specific promoter, which rate is an intrinsic property of the promoter, may limit its usefulness. Other approaches for controlling expression of therapeutic genes included the use of two-component gene switches that are activated or inactivated by a small-molecule regulator. Well-known switches are controlled by tetracycline, ecdysterone, mifepristone or rapamycin. While these switches may allow for stringent on-off regulation of gene activity, basal gene activity cannot be controlled, and intermediate levels of gene activity are typically difficult to achieve. More important, gene expression cannot be locally restricted by a small molecule regulator that readily diffuses through tissues. Because an activating heat treatment can be directed to a target tissue, heat shock gene promoters should provide an effective means for restricting expression of a gene delivered by a viral or other vector to a target tissue. These promoters have also been suggested to be useful for controlling viral replication (U.S. Ser. No. 09/850270). Some of these promoters, in particular a human hsp70B gene promoter isolated and characterized by the inventor, have minimal basal activity and a heat-induced activity that can be a thousand fold greater than basal activity. Such promoters should be particularly useful for achieving strict regulation of localized gene expression or virus replication. However, there is a downside to using promoters that are activated by transient heat and other proteotoxic stresses. After introduction into an experimental animal or a human, inadvertent activation of these promoters can occur during a fever or ischemia/reperfusion, exercise, exposure to oxidant or other proteotoxic agent, or, possibly, even hormonal stress. The confounding effects of such inadvertent, non-directed activation of virus replication or gene expression will be unacceptable in many applications. Gene switches that are only activated by a combination of heat (or other proteotoxic stress) and a small molecule regulator were previously described and were suggested to be useful for achieving safe spatial as well as temporal control of therapeutic gene expression (U.S. Pat. No. 6,342,596; U.S. patent application Ser. No. 10/046,420).

SUMMARY OF THE INVENTION

The present invention relates to a modified, conditionally replicating virus whose genome includes a gene switch that controls the expression of a viral gene required for efficient replication of the modified virus wherein the gene switch is activated in a cell infected by the modified virus upon exposure of the cell to heat and a small-molecule regulator. A modified, conditionally replicating virus of the invention is an engineered or recombinant virus whose replication is controlled by a mechanism not inherently present in the corresponding wildtype virus, which mechanism allows for deliberate induction of replication of the modified, conditionally replicating virus. A gene required for efficient replication of a virus is a viral gene that when defective reduces the rate of replication to less than about 25%, preferably less than about 10%, of the rate observed in the presence of the functional gene.

The invention similarly relates to a modified, conditionally replicating virus whose genome includes a gene switch that controls the expression of a viral gene required for efficient replication of the modified virus, wherein in a cell infected by the modified virus the gene switch can be activated by exposure of the cell to heat in the absence of a small-molecule regulator of the gene switch. More than one viral gene required for efficient replication may be placed under the control of the gene switch present in the modified, conditionally replicating viruses of the invention. A modified, conditionally replicating virus of the invention may further include a passenger gene. Expression of such a passenger gene can also be regulated by the gene switch that controls viral replication. Preferably, a modified, conditionally replicating virus is derived from a member of the Adenoviridae, Herpesviridae, and Retroviridae families. More preferably, a modified, conditionally replicating virus is derived from a member of the Adenoviridae family. Most preferably it is derived from a virus of the genus Adenovirus. If the modified, conditionally replicating virus is an adenovirus, the genes that can be controlled by the gene switch include the genes encoding E1A, E1B and E4.

The gene switch that controls the expression of one or more viral genes, whose products are required for efficient viral replication, minimally consists of two components. The first component is a gene for a transactivator that is activated or inhibited by a small-molecule regulator. In preferred embodiments, expression of the transactivator gene is controlled by a functionally linked nucleic acid sequence that acts as a heat shock promoter and is activated by transient heat or a transient proteotoxic stress through the intermediary of endogenous heat shock factor 1 (HSF1), or by a nucleic acid sequence containing a tandem or hybrid promoter that acts as a heat shock promoter as well as a transactivator-responsive promoter and is activated by transient heat or proteotoxic stress through the intermediary of endogenous HSF1 and active transactivator. The term “transactivator-responsive promoter” as used herein means a promoter that activates transcription of a functionally linked gene subsequent to binding of activated transactivator. Depending on the choice of transactivator, the DNA-binding activity or transcriptional activity of a transactivator may be induced or inhibited upon interaction of the transactivator with its small-molecule regulator or ligand. It is noted that the above single first component may be replaced by a set of two or more components that together perform the function of the single first component. For example, a transactivator gene that is functionally linked to a nucleic acid sequence which acts as a heat- and transactivator-responsive promoter may be replaced by a set of two genes encoding the same transactivator, the first gene being functionally linked to a nucleic acid that acts as a heat shock promoter and the second gene to a nucleic acid that acts as a transactivator-responsive promoter. Further, a transactivator may be a hetero-oligomeric protein. In such a case, the first component of a gene switch would the an assembly of all required subunit genes, at least one of which genes would be functionally linked to a nucleic acid sequence which acts as a heat shock promoter or as a heat-activated and transactivator-responsive promoter. The second component of the gene switch is a nucleic acid sequence that acts as a promoter responsive to the transactivator, which promoter is functionally linked to the viral or passenger gene to be regulated. The second component may also be a set of similar nucleic acid sequences that act as transactivator-responsive promoters and are functionally linked to viral and/or passenger genes to be regulated. In alternative embodiments, the first gene switch component is a transactivator gene, or set of subunit genes for a hetero-oligomeric transactivator as the case may be, that is expressed continuously (constitutively) in a host cell. The second component can be a modified heat shock promoter (including an appropriate RNA leader region) or a set of such promoters that are activated by transient heat or other proteotoxic stress and repressed by the transactivator. Binding of a small-molecule regulator to the transactivator can, respectively, inhibit or enable its repressing function. Hence, the resulting gene switch is active in cells exposed to heat or proteotoxic stress in the presence or absence, respectively, of the small-molecule regulator. In yet other alternative embodiments, the first component is a transactivator gene, or set of subunit genes for a hetero-oligomeric transactivator as the case may be, that is expressed continuously (constitutively) in a host cell. The second component can be a modified heat shock promoter or a set of such promoters that require co-activation by transactivator and endogenous HSF1. Transactivator may be activated by a bound small-molecule regulator or, alternatively, upon removal of a bound small-molecule regulator.

The invention also relates to a conditionally replicating pair of modified viruses consisting of (a) a first virus that is defective in one or more genes required for efficient replication and whose genome includes a gene for a small-molecule-regulated transactivator, the gene for the small-molecule-regulated transactivator being functionally linked to a nucleic acid sequence that acts as a heat shock promoter, and (b) a second virus comprising all genes required for efficient replication of which genes at least one gene is under the control of a promoter that is responsive to the small molecule-regulated transactivator of the first virus and is not complemented by a functional gene of the first virus, wherein expression of all genes of the second virus required for its replication will complement the defects of the first virus and result in replication of the first virus. This embodiment of the invention relates to a pair of modified viruses whose combined genomes contain all genetic information required for conditional replication of the virus pair. The first virus is a replication-defective virus lacking one or several genes required for efficient replication of the virus, whereas the second virus contains all genes necessary for its efficient replication, but one or more of the latter genes have been placed under the control of a gene switch that is activated by a transient stress such as heat and a small-molecule regulator. The stress- and small-molecule regulator-controlled gene or genes of the second virus are genes with functions in replication that cannot be provided by functional genes present in the first virus. In a further embodiment of the modified virus pair of the invention, one or more genes required for efficient replication are not present or are nonfunctional in the second virus. The missing gene products are supplied by the first virus whose genome comprises functional versions of the genes absent or nonfunctional in the second virus.

The gene switch that confers stress (e.g., heat) and small-molecule regulation upon one or more genes of the second virus comprises minimally two components that are distributed between the two viruses. The first virus contains the transactivator component that consists of a gene for a nonviral small-molecule-regulated transactivator, or of two or more genes if the transactivator is a heterooligmeric factor. The gene or, in the case of heteromeric transactivators, at least one of the genes for the transactivator is functionally linked to a nucleic acid sequence that acts as a heat shock promoter. In a variant embodiment, the transactivator gene, or at least one of the genes encoding a subunit of a multimeric transactivator, is functionally linked to a nucleic acid sequence that acts both as a heat shock promoter and a transactivator-responsive promoter. The transactivator component may also comprise two identical transactivator genes, of which one is functionally linked to a nucleic acid sequence that acts as a heat shock promoter and the other is linked to a nucleic acid sequence that acts as a transactivator-responsive promoter. The second virus contains the second gene switch component that is a nucleic acid sequence acting as a transactivator-responsive promoter. This promoter sequence is joined functionally to a gene required for efficient replication of the second virus. Multiple genes of the second virus may be joined to such a promoter sequence, subjecting them to control by the transactivator. Transactivator may be activated by a bound small molecule regulator or, alternatively, upon removal of a bound small molecule regulator.

The invention also concerns a conditionally replicating pair of modified viruses consisting of (a) a first virus that is defective in one or more genes required for efficient replication and whose genome includes a gene for a small molecule-regulated transactivator, the gene for the small molecule-regulated transactivator being functionally linked to a nucleic acid sequence that acts as a continuously active promoter, and (b) a second virus comprising all genes required for efficient replication of which genes at least one gene is under the control of a promoter that is activated upon simultaneous binding of a heat shock factor and activated transactivator and is not complemented by a functional gene of the first virus, wherein expression of all genes of the second virus required for its replication will complement the defects of the first virus and result in replication of the first virus. The first component of the gene switch in this particular embodiment consists of a continuously expressed gene for a small-molecule-regulated transactivator that is present in the genome of the first virus and is active either in the absence or in the presence of the small-molecule regulator. The second component consists of a promoter that is used to regulate one or more genes in the second virus required for efficient replication of the virus. This promoter is only active in a cell infected with the conditionally replicating pair of modified viruses subsequent to heat exposure of the cell in the presence (in the case of a small-molecule-activated transactivator) or absence (in the case of a transactivator that is inhibited by its small-molecule ligand) of small-molecule regulator. A promoter of this type may be assembled by supplementation of a basal promoter with binding sequences for heat shock transcription factor and transactivator. Alternately, one or more binding sequences for the transactivator may be introduced in an existing heat shock promoter such as the human hsp70B promoter.

The modified viruses of the conditionally replicating virus pair are typically derived from viruses of the same family, preferably from viruses of the same genus, and most preferably from viruses of the same species. These preferences are derivative of the requirement that products of one or more genes of the second virus of a pair need to be capable of complementing genetic defects of the first virus of the pair. Occasionally, this requirement is fulfilled even though the two viruses of a pair are derived from viruses of different families. An example pair of this kind could consist of a first virus derivatized from an adeno-associated virus and a second virus from an adenovirus. A further condition that needs to be fulfilled by a conditionally replicating virus pair of the invention is that the functions of the gene or genes in the second virus that are under transactivator control cannot be provided by functional genes in the first virus.

A conditionally replicating virus pair of the invention may further include a passenger gene that is inserted into the genome of the first virus of a pair. The passenger gene may be placed under the control of the same small-molecule-regulated transactivator that controls viral replication. Preferably, the modified viruses of a pair are modified members of the Adenoviridae, Herpesviridae, and Retroviridae families, with both viruses of a pair belonging to the same family. More preferably, each virus of a pair is derived from a member of the Adenoviridae family. Most preferably, each virus of a pair is an adenovirus. If the second virus of a conditionally replicating virus pair of the invention is a modified adenovirus, the genes that can be controlled by the gene switch include the E1A, E1B and E4 genes of the second virus.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 presents a generic gene switch that can be activated by transient heat or proteotoxic stress in the absence or presence, respectively, of a small molecule regulator.

FIG. 2 provides an example of the switch of FIG. 1, in which switch generic transactivator is replaced with mifepristone-activated activator GLVP and small-molecule regulator with mifepristone.

FIG. 3 outlines in part A how the generic switch of FIG. 1 can be introduced in the genome of a single adenovirus and in parts B and C how switches of FIGS. 1 and 2 can be incorporated in the genomes of a pair of adenoviruses, respectively.

FIG. 4 presents in part A a generic gene switch that can be activated by transient heat or proteotoxic stress in the presence or absence, respectively, of a small molecule regulator. The switch contains an auto-activation loop that causes transactivator expression to be maintained subsequent to transient heat or stress activation. Part B of the Figure shows an example switch, in which generic transactivator is replaced with mifepristone-activated transactivator GLVP.

FIG. 5 outlines how an example switch of FIG. 4B can be incorporated in the genome of a single adenovirus (part A) or the genomes of a pair of adenoviruses (parts B and C), respectively. Part C of the Figure further illustrates how a passenger gene can be introduced.

FIG. 6 outlines two types of alternative gene switches that can or may be used to regulate replication of viruses of the invention as well as passenger gene expression.

FIG. 7 provides the nucleotide sequence of pShuttle (SEQ ID NO: 1).

FIG. 8 provides the nucleotide sequence of pGeneN5-His (SEQ ID NO: 2).

FIG. 9 provides the nucleotide sequence of pXC1 (SEQ ID NO: 3).

FIG. 10 provides the nucleotide sequence of pSwitch (SEQ ID NO: 4).

DETAILED DESCRIPTION

Unless otherwise defined, all terms shall have their ordinary meaning in the relevant art. The following terms are defined:

“Replication of virus” or “virus/viral replication” is understood to mean multiplication of viral particles.

A “modified, conditionally replicating virus” of the invention is an engineered or recombinant virus whose replication is controlled by a mechanism not inherently present in the corresponding wildtype virus, which mechanism allows for deliberate induction of replication of the modified, conditionally replicating virus. A gene required for efficient replication of a virus of the invention is a viral gene that when defective reduces the rate of replication to less than about 25%, preferably to less than about 10%, of the rate observed in the presence of the functional gene.

“Proteotoxic stress” is a physical or chemical insult that results in increased protein unfolding, reduces maturation of newly synthesized polypeptides or causes synthesis of proteins that are unable to fold properly.

A “small-molecule regulator” is understood to be a low molecular weight ligand of a transactivator used in connection with this invention. Depending on the transactivator concerned, the small-molecule regulator can inhibit or activate the transactivator. Small-molecule regulator is typically, but not necessarily, smaller than 1000 Dalton (1 kDa).

“Transactivator”. To be able to use a single term for all regulatory proteins used in gene switches described herein, “transactivator” shall be understood hereinafter as being a DNA-binding protein, whose binding to a specific DNA sequence can affect, positively or negatively, transcription of a nearby gene. This definition includes traditional transactivators as well as proteins that interfere with transcription.

“Activated” when used in connection with a transactivated gene means that the rate of expression of the gene is substantially greater, typically by at least one order of magnitude, after activation than before activation. When used in connection with a transactivator, “active” or “activated” refers to a DNA-binding- and transactivation-competent form of a transactivator in the case of a positively acting transactivator, and to a DNA-binding-competent form of a transactivator in the case of a negatively acting transactivator. When used in connection with a gene switch, “activated” means that the activity of the target gene of the gene switch is substantially greater, typically by at least one order of magnitude, after activation than before activation.

“Promoter of a heat shock gene”, “heat shock gene promoter” and “heat shock promoter” are used synonymously.

“Gene switch”is a molecular device that can be used to achieve regulation of a gene of interest. Minimally, a gene switch should allow an inactive gene to be activated or an active gene to be inactivated. A nucleic acid sequence functional as a regulated promoter of transcription, e.g., a heat shock promoter, represents a simple form of a gene switch. The gene switches used in connection with the invention typically consist of two types of elements, a small-molecule-regulated transactivator gene component and a target gene promoter component that is activated, co-activated or repressed by the activated or active form of the transactivator.

Herein, a virus, whose genome includes a non-viral gene, is either referred to as a “virus” or a “viral vector”.

“Recombinant” or “modified” refers to the presence of an alteration caused by an experimenter.

“Passenger gene” refers to any protein- or RNA-coding gene other than a transactivator gene that is introduced into a virus of the invention.

“Conditionally replicating pair of modified viruses” refers to two nonidentical viruses (i.e., viruses differing in their genomes) that are both capable of being replicated upon co-introduction in the same cell under certain specific conditions. When introduced in separate cells, each of the viruses of the pair replicates at a substantially reduced rate that is typically at least about an order of magnitude lower than the rate achieved in the presence of the other virus.

Preferred viruses of the present invention possess the engineered property that they only replicate when cells containing them are exposed to heat or another proteotoxic stress in the presence of a small-molecule regulator. Replication of other preferred viruses can be triggered by heat or other proteotoxic stress in the absence of a specific small molecule regulator that represses replication. The viruses can further contain a passenger gene that may or may not be activated under the same conditions. Because heat can be directed, systemically present viruses of the invention can be activated locally, i.e., in a spatially restricted manner. The viruses of the invention may be used for research purposes. They may be introduced into the germline of an animal or may be introduced systemically or locally at any stage of development, including adult stage. After administration or withdrawal, respectively, of small-molecule regulator, the experimental animal may be subjected to one or more courses of localized heat to activate virus in a particular tissue or group of cells, resulting in the ablation of the targeted cells and cells in surrounding tissue and/or in activation and local spreading of an activated passenger gene and, consequently, of the product of the passenger gene. Such experiments can assist in defining the function of particular tissues or groups of cells in an adult or developing animal or in elucidating the biological or therapeutic consequences of local expression of a passenger gene. The viruses of the invention will also be useful in cell-based therapies. Cells of heterologous origin may provoke a graft-vs-host response, in spite of immunosuppressive therapy. Conditionally replicating viruses present in these cells may be specifically activated in a tissue in which rejection is observed to achieve localized killing of offending cells while maintaining the benefit of the therapy in tissues in which no rejection is observed. In other applications, immune cells containing conditionally replicating virus of the invention may be administered as a tumor therapy. Immune cells reaching the tumor may be locally activated by heat in the systemic presence (or absence) of a small-molecule regulator to trigger virus replication and cell lysis in the tumor tissue. Local cell lysis will provoke a local inflammatory response that should enhance development of specific immunity. Alternatively, cells may be infected with virus of the invention also including a passenger gene that is a therapeutic gene. After localized or systemic administration of the cells, virus replication may be activated in a desired tissue location to trigger virus replication and local dissemination of a therapeutic gene, avoiding, e.g., toxicity associated with systemic expression of the therapeutic protein. The viruses of the invention may also be used in experimental gene therapy using model animals and, eventually, humans. For example, a virus of the invention may be introduced into a tumor. Heat treatment in the systemic presence or absence, respectively, of a small-molecule regulator will initiate virus replication and tumor cell killing. The therapeutic efficacy of virus of the invention may be enhanced by the presence of a therapeutic gene, e.g., a passenger gene encoding a cytotoxic protein. Such virus will not only be induced to replicate in tumor tissue but also to express the toxic protein, resulting in increased anti-tumor activity. Alternatively, a virus of the invention may not by itself kill cells but may include a therapeutic gene, e.g., a passenger gene encoding a cytotoxic protein. Localized replication of the virus in a tumor will result in the dissemination of virus and therapeutic gene throughout the tumor. Tumor cells will be killed by the action of the therapeutic protein.

Replication of a virus of the invention is controlled by a gene switch that is activated by heat or other proteotoxic stress and the specific presence or absence, respectively, of a small-molecule regulator. Gene switches employed in the invention contain two types of components. Preferred gene switches used in connection with the conditionally replicating viruses of the invention comprise (1) a gene for a small-molecule-activated or small molecule-inhibited transactivator that is functionally linked to a nucleic acid sequence that acts as a heat shock promoter, typically a promoter from a heat shock gene, and (2) a promoter that is responsive to the transactivator. A promoter said to be responsive to a transactivator is a promoter that is activated by the active form of the transactivator. A gene to be regulated by the switch is functionally linked to the latter promoter that is responsive to the transactivator. A gene to be regulated (i.e., a target gene) may be an E1A, E1B or E4 gene in the case of a conditionally replicating adenovirus of the invention. The operation of such a gene switch is outlined in FIG. 1.

A heat shock gene is defined herein as any gene, from any eukaryotic organism, whose activity is substantially enhanced when the cell containing the gene is exposed to a temperature above its normal growth temperature. Typically, the genes are activated when ambient temperature is raised by 3-10° C. Heat shock genes comprise genes for the “classical” heat shock proteins, i.e., Hsp110, Hsp90, Hsp70, Hsp60, Hsp40, and Hsp20-30. They also include other heat-inducible genes such as genes for MDR1, ubiquitin, FKBP52, hemoxidase and other proteins. The promoters of these genes, the “heat shock promoters”, contain characteristic sequence elements referred to as heat shock elements (HSE) that consist of perfect or imperfect sequence modules of the type NGAAN or AGAAN, which modules are arranged in alternating orientations (Xiao and Lis. 1988. Science 239, 1139-1142; Amin et al. 1988. Mol. Cell. Biol 8, 3761-3769; Fernandes et al. 1994. Nucleic Acids Res. 22, 167-173). These elements are highly conserved in all eukaryotic cells such that, e.g., a heat shock promoter from a fruit fly is functional and heat-regulated in a frog cell (Voellmy and Rungger. 1982. Proc. Natl. Acad. Sci. USA 79, 1776-1780). HSE sequences are binding sites for heat shock transcription factors (HSFs; reviewed in Wu. 1995. Annu. Rev. Cell Dev. Biol. 11, 441-469). The factor primarily responsible for activation of heat shock genes in vertebrate cells exposed to heat or a proteotoxic stress is HSF1 (Baler et al. 1993. Mol. Cell. Biol. 13, 2486-2496; McMillan et al. 1998. J. Biol. Chem. 273, 7523-7528). Preferred promoters are those from inducible hsp70 genes. A particularly preferred heat shock promoter used in the viruses of the present invention is the promoter of the human hsp70B gene (Voellmy et al. 1985. Proc. Natl. Acad. Sci. USA 82, 4949-4953). It is noted that heat shock promoters may also be prepared synthetically or semisynthetically. For example, a basal promoter sequence (containing only elements specifiying RNA polymerase II complex binding and transcription initiation) from any RNA polymerase II gene such as a viral thymidine kinase gene, an actin gene, or even a heat shock gene can be converted to a heat shock promoter by the addition of short nucleic acid elements containing a copy of the above-defined HSE sequence. Amin et al. 1988.

Examples for small-molecule-regulated transactivators than can be incorporated in the gene switches used in viruses of the invention include tetracycline/doxocycline-regulated tet-on and tet-off repressors (Gossen and Bujard. 1992. Proc. Natl. Acad. Sci. USA 89, 5547-5551; Gossen et al. 1996. Science 268, 1766-1769), ecdysterone/ponasterone-regulated insect steroid receptor-based transcription factor EcR/RXR (No et al. 1996. Proc. Natl. Acad. Sci. USA 93, 3346-3351), mifepristone-regulated GAL4-steroid receptor-activation domain chimeras such as GLVP (Wang et al. 1997. Nat. Biotechnol. 15, 239-243; Wang et al. 1997. Gene Ther. 4, 432-441), versions of which transactivator are now also commercialized under the name “GeneSwitch” (Invitrogen Corporation, Carlsbad, Calif.), and rapamycin-regulated chimeric factor NFκB-FRB/FKBP-ZFHD1 (Rivera et al. 1996. Nat. Med. 2, 1028-1032; Magari et al. 1997. J. Clin. Invest. 100, 2865-2872). Other small-molecule-regulated transactivators may be used, provided that they can be employed to control the activity of a target gene without also causing widespread deregulation of genes of the host cell and provided further that the associated small-molecule regulators have a sufficiently low toxicity for the host cell at their effective concentrations.

In the example in FIG. 2, a gene for mifepristone-activated, chimeric transactivator GLVP is functionally linked to strictly heat-regulated human hsp70B heat shock promoter. The target gene is a gene required for viral replication such as, in this example, the E1A gene of adenovirus. The target gene is functionally linked to a promoter that is responsive to the transactivator. In the present example, a suitable promoter will be a minimal promoter supplemented with binding sites for GAL4. A gene switch of the kind outlined in FIG. 1 may be incorporated in a viral vector to generate a conditionally replicating virus of the invention as is shown in FIG. 3A. In this example, a transactivator-responsive promoter replaces the E1A promoter of adenovirus, and a transactivator gene cassette replaces a non-essential gene segment of the virus, e.g., an E3 region in adenovirus. Alternatively, or if there is insufficient space in the deleted virus genome for inclusion of a chosen transactivator gene, the gene switch may be distributed between two viral vectors to generate a conditionally replicating pair of modified viruses of the invention. In the example in FIG. 3B, an hsp70B-directed transactivator gene is inserted into the genome of a typical replication-deficient adenovirus lacking E1 and, possibly, E3 sequences, and a transactivator-responsive promoter replaces the E1A promoter of an otherwise wildtype adenovirus genome. A specific example pair of conditionally replicating viruses of the invention is shown in FIG. 3C. The first virus is a replication-deficient adenovirus that minimally lacks E1A and E4 functions. An hsp70B promoter—glvp gene cassette has been inserted in the genome of this virus. The second adenovirus is wildtype (or derived from wildtype by deletion of nonessential sequences, e.g., sequences from the E3 region), except for the E1A and E4 promoter regions that have been replaced by transactivator-responsive promoters.

In order for replication of any of the two viruses of a conditionally replicating virus pair of the invention to occur, a cell will need to be co-infected with the recombinant virus pair. Gene switch-directed virus replication would be effected as follows: subsequent to infection of a cell with the recombinant virus combination of FIG. 3C, the cell would be exposed to an appropriate concentration of small-molecule regulator mifepristone (10⁻¹⁰ to 10⁻⁶ M in the case of a mammalian cell) and subjected to a transient heat treatment (at 41-45° C. for 15-120 min in the case of a mammalian cell) to activate the hsp70B promoter. GLVP will be expressed from the hsp70B-glvp cassette present in the genome of the first virus and be activated by mifepristone. Active GLVP will then transactivate E1A and E4 gene expression from the second virus genome, resulting in replication of the virus pair. Cells in which the adenoviral replication occurred will lyse, virus particles will be liberated, and adjacent cells will be co-infected. Upon administration of a further transient heat treatment, replication in the secondarily infected cells will be triggered, and so forth.

A conditionally replicating virus or virus pair of the invention may also include a passenger gene. Whether the passenger gene or even a transactivator gene can be incorporated into a viral genome containing all genes and other sequences required for replication will depend on the type of virus, the maximal genome size for packaging, knowledge about non-essential viral genes that can be deleted, and the sizes of transactivator and passenger genes to be inserted. In the case of adenovirus, it may be necessary to insert the passenger gene and, possibly, also the transactivator gene into a partially deleted or completely gutted viral genome. As illustrated by the example of a conditionally replicating virus pair of the invention shown in FIG. 5C, the first virus is a replication-deficient adenovirus. Space provided by sizeable deletions of viral genes, typically E1 and E3 gene regions (and in this particular case also E4 sequences), will be taken by a passenger gene and a transactivator gene. The transactivator gene, here glvp, will be under the control of a functionally linked hsp70B promoter, or the hybrid or tandem hsp70B-glvp-r promoter actually shown in the Figure (and discussed below). The passenger gene may be linked to GLVP transactivator-responsive promoter glvp-r or another promoter, as the application may require. The second virus will contain a complete adenoviral genome except for substitution of E1A and E4 promoters with glvp-r promoters.

The modified viruses of a conditionally replicating virus pair are typically derived from viruses of the same family, preferably from viruses of the same genus, and most preferably from viruses of the same species. These preferences are derivative of the requirement that products of one or more genes of the second virus of a pair need to be capable of complementing genetic defects of the first virus of the pair. Occasionally, this requirement is fulfilled even though the two viruses of a pair are derived from viruses of different families. An example pair of this kind could consist of a first virus derivatized from an adeno-associated virus and a second virus from an adenovirus. A further condition that needs to be fulfilled by a conditionally replicating virus pair of the invention is that the functions of the gene or genes of the second virus that are under transactivator control are not also provided by functional genes of the first virus.

It is noted that it is not necessary that any one of the two viruses of a conditionally replicating virus pair of the invention contain all genes required for replication. The genes may be distributed between both viruses. What is critical is that, together, the two viral genomes encode all required viral functions and that the expression of at least one viral gene product required for efficient replication of both viruses is placed under the control of a heat-activated and small-molecule-regulator-dependent gene switch.

Most preferably, replication of viruses and virus pairs of the invention and, if desired, of passenger genes, is controlled by more complex switches of the kind described in U.S. Pat. No. 6,342,596 and in U.S. patent application Ser. No. 10/996,420. Unlike the above-described switches, these switches additionally contain an auto-activating element that allows them to remain active long after an activating heat treatment or exposure to other proteotoxic stress. A generic outline of the components and operation of such switches is shown in FIG. 4A. The switch comprises two types of components. The first component is a gene for a small-molecule-regulated transactivator, which gene is functionally linked to a nucleic acid sequence that acts as a heat shock promoter as well as a transactivator-responsive promoter. This nucleic acid sequence may contain a hybrid promoter comprising binding sites for both HSF1 and transactivator. Alternatively, it may consist of a tandem assembly of two separate promoters, one of them a heat shock promoter and the other a transactivator-responsive promoter, provided that each of the promoters can direct expression from the linked transactivator gene. The second component is a transactivator-responsive promoter to which a target gene is functionally linked. When a cell containing the two types of switch components is exposed to transient heat or other proteotoxic stress, (endogenous) HSF1 is activated and induces transcription from the transactivator gene. Depending on whether the particular transactivator used is activated or inhibited by a small-molecule regulator, newly made transactivator will activate the expression of the target gene as well as promote expression of additional transactivator protein in the presence or absence of the small-molecule regulator. As a consequence, transactivator level is maintained even after HSF1 has been inactivated subsequent to the transient stress, and target gene expression continues. Removal or addition, respectively, of small-molecule regulator will result in the inactivation of the gene switch. FIG. 4B shows an example of a gene switch of this kind, in which gene switch the transactivator is mifepristone-activated transactivator GLVP and the target gene is the E1A gene of an adenovirus. In FIG. 5, the incorporation of the latter example switch in conditionally replicating adenoviruses of the invention is illustrated. FIG. 5A illustrates how the glvp gene (“transactivator gene”) and the linked hybrid or tandem promoter (“hsp70B-glvp-r”) are introduced into the E3 region subsequent to deletion of sequences from this region. The E1A promoter is replaced by GAL4-binding site-containing promoter glvp-r that is responsive to GLVP. Whether such a recombinant can actually be made depends on the virus' packaging limit and on the lengths of the deleted and inserted sequences, respectively. In the example in FIG. 5B, the same gene switch is distributed over a pair of viruses. The first virus (bottom) is a replication-deficient adenovirus that may lack E1, E3 and E4 sequences but contain the glvp gene and the linked hybrid or tandem promoter that is activated by heat and active GLVP. The second virus (top) may contain a complete adenoviral genome, except for replacement of E1A and E4 promoters with glvp-r promoters. As shown in the graph in FIG. 5C, a passenger gene may also be included in the defective genome of the first virus. Expression of the passenger gene may be controlled by a transactivator-responsive promoter or another promoter.

Alternatively, replication of a conditionally replicating virus of the invention and, if desired, expression of a passenger gene can be controlled by a type of gene switch (FIG. 6A) that comprises (1) a continuously or constitutively expressed gene for a transactivator (that is not transactivation-competent), whose DNA-binding activity is regulated by binding of a small-molecule regulator and (2) a nucleic acid sequence that acts as a heat shock promoter and may also include 5′untranslated sequences, which nucleic acid sequence is modified to contain one or more transactivator binding sites. The latter promoter is functionally linked to a viral or passenger gene to be regulated. Transactivator binding sites are placed in the heat shock promoter (and/or RNA leader region) in such a way that binding of transactivator prevents heat-activated transcription of the linked gene. Binding of transactivator to sites inserted near HSF1 binding sites may prevent HSF1 binding to the promoter, and binding of transactivator to sites inserted in the 5′ untranslated sequences may inhibit transcription elongation. The resulting gene switch is activated by transient heat in the presence or absence, respectively, of small molecule regulator.

An additional type of gene switch (FIG. 6B) that may be used to control replication of conditionally replicating viruses and virus pairs of the invention and, if desired, expression of passenger genes comprises a constitutively expressed gene for a transactivator, whose DNA-binding activity (or, possibly, transcriptional competence) is activated or inhibited by binding of a small molecule regulator, and a modified heat shock promoter including one or more binding sites for the transactivator. These binding sites may be low-affinity binding sites for the transactivator, or, possibly, half sites, that are insufficient to allow activation of the promoter by the transactivator alone. The promoter is effectively co-activated by HSF1 induced by a transient heat or other proteotoxic stress and active transactivator. A modified heat shock promoter that requires co-activation may be obtained by deletion of all but one heat shock elements in a heat shock promoter, whose activation depends on the presence of multiple heat shock elements (e.g., the human hsp70B promoter), and addition of one or more transactivator binding sites. A promoter of this type may also be assembled by supplementation of a basal promoter with binding sequences for heat shock transcription factor and transactivator. Alternately, one or more binding sequences for the transactivator may be introduced in an existing heat shock promoter such as the human hsp70B promoter.

Viruses particularly suitable for use in the present invention are those that were previously used as replicating or oncolytic viral vectors such as adenoviruses, herpesviruses, and retroviruses, including foamy viruses.

The biology and genetics of adenoviruses are well understood (Russell. 2000. J. Gen. Virol. 81, 2573-2604; Hitt et al. 1997. Advances in Pharmacology 40, 137-206). Persons skilled in the art know how to prepare and administer replication-competent or replication-deficient adenovirus vectors. Strategies used to control replication of adenovirus were recently reviewed (Post et al. 2003. Hum. Gene Ther. 14, 933-946). Typically, conditional replication of adenovirus was achieved by replacing the E1A, E1B or E4 viral promoter with a regulated or tissue-selective promoter of choice. In some cases, two of the latter viral promoters were replaced. For example, Hernandez-Alcoceba et al. (Hum. Gene Ther. 11, 2009-2024 (2000)) prepared a conditionally replicating adenovirus by replacement of the E1A and E4 promoters by truncated pS2 promoters containing binding sites for estrogen receptor. The recombinant virus replicated and lysed efficiently estrogen receptor-positive but not estrogen-receptor-negative cells. Yu et al. (Cancer Res. 59, 1498-1504 (1999)) constructed conditionally replicating adenovirus 764 by substituting the E1A and E1B promoters with a PSE (prostate-specific enhancer) promoter and a promoter fragment from a kallikrein 2 gene, respectively. Virus 764 efficiently replicated in and killed cells from a prostate tumor cell line but not cells from several other cell lines.

Herpesviruses are enveloped, double-stranded DNA viruses with large genomes (152 kbp for HSV-1). This large genome size had hampered genetic analyses and experiments in the past. This problem has been solved by the use of bacterial artificial chromosomes (BACs) that allow integration of an entire herpesvirus genome, enabling the use of efficient mutagenesis and recombination protocols to manipulate the viral genome (Wagner et al. 2002. Trends in Microbiol. 10, 318-324). The products of at least two of the immediate early genes of herpesvirus, ICP4 and ICP27, are essential for viral replication (Burton et al. 2002. Curr. Opin. Biotechnol. 13, 424-428, and references cited therein). Manipulations that place one of these genes under the control of a regulated or tissue-selective promoter will produce a conditionally replicating herpesvirus. For example, Miyatake et al. (J. Virol. 71, 5124-5132 (1997)) made use of HSV-1 mutant d120 that lacked both copies of the a4 gene, the gene encoding ICP4, and inserted into this mutant an a4 gene copy that was functionally linked to a promoter containing an albumin enhancer and promoter element. The resulting recombinant herpesvirus replicated three log scales better in albumin-expressing cells than in other cells. Furthermore, the virus effectively inhibited growth of an albumin-expressing tumor xenograft. Herpesviruses can be made to preferentially replicate in mitotically active cells by deletion of genes for thymidine kinase, ICP6 or ICP34.5 (Harrington et al. 2002. Clinical Oncology 14, 3-16, and references cited therein).

Retroviruses are single-stranded, diploid RNA viruses that have been intensively investigated and used as replication-defective vectors to transfer therapeutic genes (Vile and Russell. 1995. Br. Med. Bull. 51, 12-30). Persons skilled in the art know how to manipulate retroviral genomes and how to manufacture retroviral vectors. Preparation of retroviral vectors and their uses are described in many publications including European patent application EP 0178220; U.S. Pat. No. 4,405,712; Gilboa. 1986. Biotechniques 4, 504-512; Mann et al. 1983. Cell 33,153-159; Cone and Mulligan. 1984. Proc. Natl. Acad. Sci. USA 81, 6349-6353; Eglitis et al. 1988. Biotechniques 6, 608-614; Miller et al. 1989. Biotechniques 7, 981-990; Miller. 1992. Curr. Top. Microbiol. Immunol. 158, 1-24; PCT application No. WO 92/07943 entitled “Retroviral Vectors Useful in Gene Therapy”; Hu and Pathak. 2000. Pharmacol. Rev. 52, 493-511. Promoters driving the expression of viral genes reside in the U5 and U3 regions of provirus. The actual viral genome lacks the U5 region. Hence, it only contains the promoter of the U3 region. Recently, Logg et al. (J. Virol. 76, 12783-12791 (2002)) demonstrated that replacement of the promiscuous transcriptional control elements within the U3 region in murine leukemia virus with sequences from the promoter of the androgen-regulated probasin gene that is active in the prostate, or from a variant promoter derived from the probasin promoter, resulted in recombinant virus that preferentially replicated in prostate-derived cells. Nestler et al. (Gene Ther. 4, 1270-1277 (1997) reported the construction of a replication-competent foamy virus that lacked a large segment of the U3 region and most of the bet gene. Genes encoding prodrug-activating enzymes, i.e., herpes thymidine kinase, cytosine deaminase and polynucleoside phosphorylase were fused to the truncated bet gene. The latter “suicide” proteins were expressed as fusion proteins, which were processed to yield unfused proteins due to the presence of a self-cleaving sequence at the fusion point.

More generally, viruses suitable for use in the invention include retroviruses, and single-stranded and double-stranded DNA viruses that utilize the host transcriptional machinery and whose replication depends on proteins expressed post infection. The latter DNA viruses include the Hepadnaviridae, Parvoviridae, Papovaviridae, Circoviridae, Adenoviridae and Herpesviridae families.

Papovaviridae include at least nine human papilloma viruses and polyomavirus BK and JC.

Retroviridae include the viruses of genera Lentivirus, Spornavirus and others such as HTLV-I, HTLV-II, HIV-I, HIV-II, bovine leukosis virus, feline sarcoma and leukemia viruses, avian reticuloendotheliosis, mouse mammary tumor virus, visna viruses, equine infectious anemia virus, FIV, bovine lentivirus, SUV, and foamy virus.

Hepadnaviridae include hepatitis B-like viruses, e.g. hepatitis B and other related viruses.

Parvoviridae include the genera parvovirus, dependovirus and densovirus. Adeno-associated viruses are also members of the family.

Adenoviridae include the viruses of genera Mastadenovirus and Aviadenovirus, such as human and animal mastadenovirus strains.

Herpesviridae include the viruses of genera Simplexvirus, Varicellavirus, Betaherpesvirinae, Cytomegalovirus, lymphocryptovirus, Marek's disease-like viruses and Rhadinovirus. Examples are herpes simplex virus, varicella-zoster virus, human cytomegalovirus, Marek's disease virus, EB virus and others.

Circoviridae include the genus Circovirus. Example circoviruses are chicken anemia virus, psittacine beak and feather disease virus, and porcine circovirus.

Extensive descriptions of the biology, biochemistry and genetics of the above virus families and their known members are found, e.g., in “Virology” (B. N. Fields, D. M. Knipe, and P. M. Howley, eds.), vol. 1 and 2, Lippincott-Raven Publishers, Philadelphia, Pa. (1996).

Passenger genes may include cell-affecting genes such as genes that encode apoptopic inducers, genes that affect cell death, aging, division and DNA synthesis, mitochrondial genes, peroxisomal genes, immune response-related genes, ATP-binding proteins, cytoskeletal genes, all rescue genes, genes involved in cell damage and repair. A listing of potential yeast and mammalian genes that may be included in the viral vectors of the invention is provided below.

Yeast Genes: CELL RESCUE, DEFENSE, CELL DEATH AND AGING PRE3, PRE1, PUP2, RPN12, RPT1, MAG1, OGG1, SED1, ATH1, SPE2, GRE3, TPS2, TPS1, ATR1, ATX1, SK13, SK12, SK18, APN1, HPR5, ERG5, CCZ1, SRA1, SNF1, YCK1, YCK2, HRR25, CTA1, CTT1, WSC4, PAM1, TIR2, TIR1, HDF2, TFB4, RAD1, HAM1, LYS7, SOD1, KIN28, DIT2, ERG11, CYC7, CCP1, PHR1, DAK2, DAK1, ALR1, ALR2, HOR2, RAD17, DDC1, DDR2, ALK1, HEL1, SSL2, RAD5, SGS1, PIF1, RAD3, CDC9, REV7, NTG1, RAD18, RAD57, RAD55, XRS2, RAD30, MMS21, RAD51, RAD10, PS02, REV1, DIN7, RAD54, CDC2, PES4, POL2, REV3, RPB7, RPB4, SGE1, UBA1, UBC4, UBC5, RAD6, QR18, RNC1, NTG2, ERC1, RAD4, ETH1, FKB2, YHB1, FLR1, MEC3, ZWF1, GSH1, GRX1, TTR1, HYR1, GLR1, YCF1, FPS1, GPD1, RAS2, RAS1, CUPS, HSP26, HSP30, HSP12, HSP104, DDR48, HSC82, HSP82, MDJ1, MDJ2, HSP60, HSP78, ECM10, SSE1, SSA1, SSA3, SSA4, SSA2, SSE2, HSF1, HIG1, HDF1, HMS2, GRE1, DD11, RTA1, SIMI, LAG2, ZDS1, MET18, SNG1, NCA3, KTI12, UTH1, SUN4, SSU81, SSD1, TH14, KAR3, LIF1, SFA1, LAG1, LTV1, MDR1, SSK22, SSK2, HOL1, CIS3, HSP150, PIR3, MAC1, CUP1 A, CUP1 B, YDJ1, SSQ1, SSC1, IMP2, MPT5, ATX2, SNO2, MLP1, NHX1, NCP1, NSR1, SNF4, RAD16, RAD7, RAD14, RAD23, ROD1, MGT1, OSM1, SIP18, SAT2, MNR2, MMS2, PNT1, CYP2, PAD1, PDR5, PDR3, PDR6, RTS1, PA13, HOR7, DUN1, IRE1, MKK2, MET22, PPZ2, PTC1, PTP2, MMS4, RAD52, PDR13 SLG1, GRR1 HIT1, RDH54 BR01, PIR1 MSRA, RNR4 RNR3, HAL1, YGP1, CDC55, PPZ1, PKC1, HAL5, MKK1, HOG1, SLT2, BCKi, RAD53, SIR4, SIR3, SIR2, MGA1, FUN30, YR02, DNL4, RRD1, SAT4, RAD27, MSN2, ST11, PAU3, PAU2, PAU5, PAU1, PAU4, PAU6, (MLP1), RAD2, FZ_F1, SSU1, SOD2, CRS5, BCK2, ASM4, TIP1, TFB1, CCL1, SSL1, TFB3, TFB2, TSAI, TRX1, TRX2, ROX3, PDR1, GTS1, MCM1, SKN7, CAD 1, MSN4, YAP1, SLN1, SSK1, PBS2, UB14, RSP5, SVS1, ZRC1.

CELL GROWTH, CELL DIVISION AND DNA SYNTHESIS GSC2, PLC1, PRE3, PRE2, PRE1, PUP2, RPN12, RPT6, RPT1, DIS3, RP SOA, AGA1, AGA2, ASG7, ACH1, ACT1, SAC6, ARP100, ABP1, PAN1, ARP 2, AREIARE2, SPE2, CYR1, SRV2, ADK2, GCS1, SOH1, TUB1, TUB3 SAG1, AKR1, YAR1, SK18, ARG82, ABF1, STE6, BAR1, 8011, TUB2, RBI-2, BIG1, BI M1, BAT1, BEM1, BEM4, SBE2, BN14, BUD6, 8012, BUD9, BUD4, BUD8, RC K2, CMK1, CNA1, CMP2, CNB1, CCH1, CMD1, SRA1, YCK1, YCK2, HRR2 5, CKA2, CKA1, YCK3, EST2, TFS1, SCM4, GIC2, GIC1, CAK1, BUB2, B UB3, ESR1, RAD24, DBF20, PDS1, HPC2, NUD1, CDC47, CDC10, CDC13, C DC37, CDC1, CDC40, CDC4, CDC20, CDC6, CDC46, CDC3, KAR1, BB P1, CDC50, FUS1, KRE9, EGT2, ARP1, CHS1, CHS2, CHS3, CHS5, MS11, CAC2, R LF2, CHL4, SMC1, SMC2, CIN1, SNF7, CLC1, COF1, PAM1, LAS17, HDF2, SEC3, SNF2, SWI1, SNF5, SNF1 1, DOC1, APC2, APC5, TAP42, CDC53, KAR 9, CCE1, CLB6, CLB5, CLN3, PCL2, CLN1, PCL1, CLN2, CI-131, CLB 4, CI-132, FAR1, CKS1, CDC28, PH085, KIN28, SSN3, CLG1, DIT2, SLA1, SLA2, SP020, DPP1, RAD17, DDC1, HEL1, DNA2, RAD5, SGS1, HCS 1, PIF1, CDC9, MSH3, MSH6, MLH1, PMS1, MSH2, MSH1, POL4, REV 7, MRE11, RAD26, RAD9, RAD18, RAD57, RAD55, XRS2, MMS21, RAD51, RADIO, RAD 50, RFA3, RFA2, RFA1, RFC4, RFC5, RFC3, RFC2, RFC1, FOB1, TOP1, TO P2, TOP3, RAP1, RAD54, PR12, PR11, POL1, POI-12, CTF4, HUS2, CDC2, P ES4, POL2, DPB2, DPB3, MIP1, REV3, SSN8, GAL11, RGR1, SRB6, RP041, SEC59, DIP2, CDC14, MSG5, DYN1, UBC4, UBC9, CDC34, UBC5, UBC 1, UBC6, RAD6, QR18, ELC1, RNC1, CTS1, KEX2, APG1, SSP1, SUP35, EXM2, S PR1, EXG1, EXG2, DHS1, CAP1, CAP2, BRN1, GPR1, GIF1, MEC3, TU B4, CIS2, LTE1, SDC25, SRM1, CDC25, ROM2, BUDS, ROM1, SPT16, CDC43, G IP1, SIN4, SNF6, KRE6, GFA1, NGR1, WH12, RSR1, CIN4, RAS2, RAS1, GP A1, STE4, STE18, CDC42, MDG1, SEC4, TEM1, RH03, RH04, RI-102, RHO 1, CDC24, BEM2, BUD2, BEM3, LRG1, GPA2, SIS1, HSP82, HSF1, ABF2, HDF1, HDR1, RPD3, HSL7, HO, SBA1, HPR1, IDS2, NFI1, CSE2, MDM 1, MI-1 131, MIDI, SIMI, HIR3, SIS2, MAKI 1, LAS1, SPA2, WH14, ECM33, SET1, CTF19, CIN2, MCM16, SLK19, CYK2, CNM67, SST2, DPB11, DOS2, D FG16, AFR1, ZDS1, SR07, PEA2, FAR3, SMP2, WH13, CDC5, MET30, SAS2, SCC2, CIS 1, STN1, UTH1, PAC2, SSD1, SRP1, KRE5, KIP1, CIN8, SMY1, KIP2, KAR3, KIP3, CBF1, CBF2, SKP1, CEP3, CTF13, DBR1, LAG1, MIH1, BFR1, DIG2, DIG1, MFA1, MFA2, MFAlpha1, MFAIpha2, MID2, SSF1, MATALPHA2, MATA LPHA1, ALPHAI, ALPHA2, A2, A1, SAN1, PGD1, SPO11, MSH5, DMC 1, ISC10, M5H4, SP013, NDT80, REC104, HOPI, RED1, SP07, MUM2, ME15, S AE2, NAM8, REC107, REC102, REC114, MER1, RIM01, NDJ1, CDC54, CP R7, SY1, MCM2, CIS3, HSP150, ACE2, CDC48, ASE1, YTM1, HSM3, YD J1, ERV1, FUS3, JNM1, MCD1, MMC1, MSB1, MSB2, MPT5, ZDS2, MSN5, KEM1, MLC1, MY02, MY04, MY05, MY03, MY01, DEC1, PMD1, M DS3, ASH1, UME1, UME6, NHP6A, RFT1, TRF5, NNF1, NDC1, BIK1, KAR2, KAR5, NUM1, CDC39, MAK16, NAP1, RAD16, RAD23, NBP35, ORC1, ORC6, ORC5, ORC4, ORC3, RRR1, SIC 1, BUD3, PWP2, STE3, STE2, OPY2, STE50, STE5, PEL1, TOR1, TOR2, PIK1, STT4, MSS4, SP014, POL32, IME4, SHP1, PDS5, FEN1, CSE1, FL08, PFY1, PHB2, PHB1, POI-30, AXL1, STE23, RAD28, CDC7, SMP3, MKK2, CDC15, ARD1, CHU, PPH3, PPH21, PPH22, PTC1, SE C9, PPS1, PTP3, YVH1, PTP2, PUS4, PCH2, PCH1, CBF5, SEF1, MMS4, SHR5, RAD59, RAD52, RHC18, RGP1, RVS167, RIM9, BNR11, BN11, SPT3, SOK2, KAR 4, DBF4, SDS22, MCM3, CTF18, SR04, SPH1, FUS2, MOB1, FL08, FIG1, FIG2, END3, DFG5, CTR9, TOM1, POP2, GRR1, SCP160, SUR1, MUM3, ZIP2 CDC45, RDH54, SHE3, SHE2, SHE4, GPI1, MIF2, ESP1, HOP2, DNA43, SMC3, PAC11, PAC10, RD11, RGA1, RNR1, RNR2, RNR4, RNR3, PRPS 1, RPL10, RPS1A, MTF1, SN12, CDC12, CDC11, SPR28, CDC55, GLC7, PKC1, GI N4, SPS1, RCK1, BUB1, IME2, YAK1, YPK2, RIM11, CLA4, MKK1, MEK1, I PL1, SGV1, SLT2, KSS1, BCK1, STE11, STE20, DBF2, HSL1, NRK1, SIT4, T PD3, ELM1, MCK1, RAD53, STE7, SWE1, MPS1, SAS3, HST1, SIR4, SIR3, SIR1, SIR2, CTH1, DOM34, HST4, RVS161, DNL4, IQG1, FUN16, HYM1, RT S2, MNN10, PRK1, MCM6, SAP155, SAP4, SAP190, SAP185, MUD13, M AD1, CIK1, NUF1, SPC97, SPC42, SPC98, CDC31, NUF2, MAD3, MAD2, DI T1, YSW1, SP012, SP016, MCD4, BDF1, SGA1, GSG1, SHC1, CDA1, CD A2, SMK1, SPS2, SPR6, SLZ1, SPS4, SPR3, SPS100, SPS18, RAD27, SNZ 1, SUR4, ST11, SBE22, CSE4, BMH 1, SVL3, SCH9, (MLP1), SSF2, RAD2, CDH1, CDC27, CDC26, CDC23, CDC16, APC1, APC 11, APC4, APC9, SAP30, RSC6, RSC8, STH1, SFH1, SAS5, JSN1, BMH2, SMT4, BCK2, HOC1, ZIP1, UFE1, EST1, TEL1, ANC1, CCL1, DST1, TRX1, TRX2, TRF4, PAT1, SPT4, SP T6, CDC36, SWI5, SWI4, PHD1, SWI6, GTS1, MCM1, IME1, SKN7, MBP1, SW13, SIN3, STE12, CIN5, SDS3, SP01, MOT2, RPG1, PRT1, CDC33, TPM1, TPM2, TWF1, TEC1, TTP1, STE13, PRP8, UB14, DSK2, RSP5, D OA4, UNG1, VPS45, VAN1, VRP1, DFG10, YHM2, GL′Q3, SFP1, STE24, RME1, SAE3, ME14, NHP6B, MOB2, EST3, RIM1.

HEAT SHOCK PROTEINS CAT5, CPH1, CTT1, CYP2, DDR2, FPR2, HSC82, HSP104, HSP12, HSP150, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, KAR2, MDJ1, SIS1, S OD2, SSA1, SSA2, SSA3, SSA4, SSB1, SSB2, SSC1, SSE1, SSE2, ST11, TIP 1, TPS2, UB14, YDJ1.

MITOCHONDRIAL AAC1, AAC3, AAT1, ABC1, ABF2, AC01, ACR1, ADH3, ADK2, AEP2, AFG3, ALD1, ALD2, ARG11, ARG2, ARG5,6, ARG7, ARG8, ARH 1, AT M 1, ATP1, ATP10, ATP11, ATP12, ATP14, ATP15, ATP16, ATP2, ATP3, ATP4, A TP5, ATP6, ATP7, ATP8, ATP9, BAT1, BCS1, CBP1, CBP2, CBP3, CBP4, CB P6, CBR1, CBS1, CBS2, CCA1, CCE1, CCP1, CEM1, CIT1, CIT3, COB, CO Q1, COQ2, COQ3, COQ6, COR1, COT1, COX1, COX10, COX11, COX12, C 0X3, COX14, COX15, COX17, COX2, COX3, COX4, COX5A, COX5B, COX6, COX7, COX8, COX9, CPR3, CTP1, CYB2, CYC1, CYC2, CYC3, CYC7, CYT1, CYT2, DB156, DLD1, DTP, ENS2, ERV1, FLX1, FUM1, GCV1, GCV3, GI-04, GPD2, GSD2, GUT2, HEM1, HEM15, HSP10, HSP60, HSP78, HTS1, IDH1, ID H2, IDP1, IFM1, ILV1, ILV2, ILV3, ILV5, ILV6, IMP1, IMP2, INH1, ISM1, KG D1, KGD2, LAT1, LEU4, LIP5, LPD1, LYS12, LYS4, MAE1, MAM33, MAS1, MAS2, MBA1, MCR1, MDH1, MDJ1, MDJ2, MDM10, MDM12, MEF1, MEF2, MET13, MGE1, MGM101, MIP1, MIR1, MIS1, MMM1, MMTi, M MT2, MOD5, MOL1, MRF1, MRP1, MRP13, MRP17, MRP2, MRP20, MRP21, MRP4, MRP49, MRP51, MRP8, MRPL10, MRPL 11, MRPL13, MRPL15, MRPL16, MRPL 17, MRPL 19, MRPL2, MRPL20, MRPL23, MRPL24, MRPL25, MRPL27, MRPL28, MRP L3, MRPL31, MRPL32, MRPL33, MRPL35, MRPL36, MRPL37, MRPL38, MR PI-39, MRPL4, MRPL40, MRPL44, MRPL49, MRPL6, MRPL7, MRPL8, M RPL9, MRPS28, MRPS5, MRPS9, MRS1, MRS11, MRS2, MRS3, MRS4, MRS5, MSD1, MSE1, MSF1, MSH1, MSK1, MSM1, MSP1, MSR1, MS S1, MSS116, MSS18, MSS51, MST1, MSU1, MSW1, MSY1, MTF1, M T01, NAM1, NAM2, NAM9, ND11, NHX1, NUC1, OM45, ORFA04514, OSM1, OXA1, PDA1, PDB1, PDX1, PEL1, PET111, PET112, PET117, PET122, PET123, PET127, PET130, PET191, PET309, PET494, PET54, PET56, PET9, PETCR46, PI-1131, PHB2, PIF1, PIM1, POR1, POR2, PPA2, PSD1, PUT1, PUT2, QCR10, QCR2, QCR6, QCR7, QCR8, QCR9, RCAi, RF2, RIM 1, RIM2, RIP1, RML2, RNA12, RPM2, RP 041, SC01, SCO2, SDI-11, SDH2, SDI-13, SDI-14, SECY, SHM1, SHY1, SLS1, SMF2, SOD2, SOM1, SSC1, SS.COPYRGT.1, STF1, STF2, SUN4, SUV3, TIM17, TIM22, TIM23 TIM44, TIM54, TOM20, TOM22, TOM37, TOM40, TOM6, TOM7, TOM 70, TOM72, TRM1, TUF1, UNG1, VAR1, YAH1, YAL011W, YAT1, YBL013 W, YCR024C, YDR041W, YDR115W, YDR116C, YER073W, YFH1, YGL068W, YGR257C, YHM1, YHR075C, YHR148W, YJL200C, YJR113C, YKLO55C, YKL120W, YKL134C, YKL192C, YLR168C, YMC1, YMC2, YML025C, YMR188C, YMR31, YNL081 C, YNL306W, YNR036C, YNR037C, YOR221 C, YPL013C, ETF-BETA.

PEROXISOMAL CAT2, CIT2, CTA1, DAL7, EHD1, EHD2, FAA2, FAT2, FOX2, ICU, IDP 3, MDH3, MLS1, PEX11, PEX12, PEX13, PEX14, PEX17, PEX2, PEX3, PE X4, PEX6, PEX7, PEXB, POT1, PDX1, PXA1, PXA2, SPS19, YBR204C, YDR 449C, YHR180W DNA-ASSOCIATED A1, A2, ABF1, ABF2, ADA2, ADE12, ADR1, ALPHA1, ALPHA2, ANC1, APN1, ARGR1, ARGR2, ARGR3, ARR1, ASH1, AZF1, BAS 1, BDF1, BR F1, BUR6, CAC2, CAD1, CAF17, CATB, CBF1, CBF2, CCE1, CCR4, CDC13, CDC36, CDC39, CDC46, CDC47, CDC54, CDC6, CDC7, CDC73, CDC9, CEF1, CEP3, CHA4, CHD1, CHU, CRL4, CRZ1, CSE1, CSE2, CSE4, CTF13, CUP2, CUP9, DAL80, DAL81, DAL82, DAT1, DBF4, DMC1, DNA2, DNA43, DNL4, D OS2, DOT6, DP131 1, DPB2, DPB3, DST1, ECM22, ENS2, EST1, EZL1, FCP1, FHL1, FKH1, FKH2, FL08, FZF1, GAL11, GAL4, GAT1, GBP2, GCN4, GCNS, GCR1, GCR2, GLN3, GL03, GTS1, GZF3, HAC1, HAP1, HAP2, HAP3, HAP4, HCM1, HDA1, HDF1, HFM1, HHF1, HHF2, HH01, HHT1, HHT2, HM01, HMS1, HMS2, H0, HOP1, HPR1, HPRS, HSF1, HTA1, HTA2, HTA3, HTB1, HT 62, IFH1, IME1, IME4, INO2, IN04, IXR1, KAR4, LEU3, LYS14, LYS20, LYS21, MAC 1, MAGI, MAL13, MAL23, MAL33, MATALPHA1, MATALPHA2, MBP1, MCD1, MCM1, MCM2, MCM3, MCM6, MED6, MER2, MET18, MET28, MET30, MET31, MET32, MET4, MGA2, MGT1, MIF2, MIG1, MIG2, MIP1, MLH 1, MOL1, MOT1, MPT4, MRE11, MSH1, MSH2, MSH3, MSH4, MSHS, MS11, MSN1, MSN2, MSN4, MTF1, NBN1, NC132, NDJ1, NGG1, NHP2, NHP6 A, NHP6B, NOT3, NUC2, OAF1, OP11, ORC1, ORC2, ORC3, ORC4, ORCS, ORC6, PAF1, PCH1, PCH2, PDR1, PDR3, PGD1, PHD1, PH02, PH04, PHR1, PIF1, PIP2, PMS1, POB1, POL1, POL12, POL2, POL3, POL30, PO L4, POP2, PPR1, PRI1, PR12, PS02, PUT3, RAD1, RAD10, RAD14, RAD 16, RAD18, RAD2, RAD23, RAD26, RAD27, RAD3, RAD4, RADS, RAD50, RAD51, RAD52, RAD54 RAD55, RAD57, RAD6, RAD7, RAP1, RAT-1, RCS1, REB1, REC102, RE C104, REC114, RED1, REG1, RET1, REV3, RFA1, RFA2, RFA3, RFC1, RFC2, RFC3, RFC4, RFCS, RGM1, RGT1, RIF1, RIF2, RIM1, RIM101, RLF2, RLM1, R ME1, RMS1, ROX1, ROX3, RPA12, RPA135, RPA14, RPA190, RPA34, RPA43, RPA49, RP131 0, RPB11, RPB2, RP133, RP134, RPBS, RPB6, RPB7, RPBB, RPB9, RPC10, RPC19, RPC25, RPC31, RPC34, RPC40, RPC53, RPC82, RPD3, RP021, RP031, RP041, RRN10, RRN11, RRN3, RRNS, RRN6, RRN7, RRN9, RSC4, RSC 6, RSC8, RTG1, RTG3, SASS, SEF1, SET1, SFH1, SFL1, SGS1, SIG1, SIN 3, SIN4, SIP2, SIP4, SIR1, SIR2, SIR3, SIR4, SKN7, SK01, SMC1, SMC 2, SMP1, SNF2, SNFS, SNF6, SOK2, SPKi, SPOL, SPS18, SPT10, SPT 15, SPT16, SPT2, SPT21, SPT23, SPT3, SPT4, SPT5, SPT6, SPTB, SRI 32, SRB4, SRBS, S RB6, SRB7, SR138, SR139, SSL2, SSN3, SSN6, SSNB, SSU72, STB4, STBS, S TE12, STH1, SUA7, SWI1, SW13, SW14, SW16, SWP73, TAF19, TAF25, TBF1, TEA1, TEC1, TFA1, TFA2, TF131, TF132, TF133, TFB4, TFC1, TFC2, TF C3, TFC4, TFCS, TFG1, TFG2, TH12, TOA1, TOA2, TOP1, TOP2, TOP3, TRF4, TS P1, TUP1, TYE7, UGA3, UME6, UNGI, USV1, XRS2, YAL019W, YAP1, YA P3, YAPS, YBL054W, YBR026C, YBR150C, YBR239C, YCR106W, YDR026 C, YDR060W, YDR213W, YER045C, YER184C, YFL052W, YIL036W, YIL 130W, YJL103C, YM206C, YKL005C, YKL222C, YKR064W, YLL054C, YLRO 87C, YLR266C, YNL206C, YOL089C, YOR172W, YOR380W, YOX1, YPL133C, YPR008W, YPR196W, YRR1, ZAP1, ZIP1, ZU01.

IMMUNOSUPPRESSANT FEN1, SSH4, SHR3 CYCLINS CCU, CLB1, CLB2, CLB3, CL134, CLBS, CLB6, CLG1, CLN1, CLN2, CLN 3, CTK2, PCL1, PCL10, PCL2, PCLS, PCL6, PCL7, PCLB, PCL9, PH080, S SNB, YBR095C.

ATP-BINDING CASSETTE PROTEINS ADP1, ATM 1, CAF16, GCN20, MDL1, MDL2, PDR10, PDR11, PDR12, PDR15, PDRS, PXA1, PXA2, SNQ2, STE6, YBT1, YCF1, YDL223C, YD R091C, YEF3B, YER036C, YHL035C, YKR103W, YKR104W, YLL015W, YNR070W, YOR011W, YOR1, YPL226W.

CYTOSKELETAL ABP1, ACF2, ACT1, AFR1, AIP1, AIP2, ARP3, AUT2, AUT7, BEM1, BI M1, BN11, BN14, BUD3, BUD6, CAP1, CAP2, CDC10, CDC11, CDC12, CDC 3, CIN1, CIN2, CIN4, CMD1, COF1, CRN1, END3, GIC1, GIC2, GIN4, J NM1, KAR9, KIP2, KIP3, LAS 17, MDM1, MHP 1, MY01, MY02, MY03, MY04, MY05, PFY1, RVS161, RVS167, SAC6, SAC7, SEC 1, SHE3, SHM2, SLA1, SL A2, SMY1, SMY2, SPA2, SPH1, SPR28, SPR3, SRV2, TCP1, TPM1, TPM2, TUB1, TUB2, TUB3, VPS16, VRP1 APOPTOSIS ATP1, ATP14, ATP15, ATP16, ATP2, ATP3, ATP4, ATPS, ATP6, ATP7, ATP8, ATP9, CYC1, SH01, SSK2, SSK22, SW13, SXM1.

CELL RESCUE ACC1, ALD6, BCK1, BEM 1, BEM2, BIM1, BMH1, BMH2, CAN1, CBF1, CDC1, CDC14, CDC15, CDC20, CDC25, CDC28, CDC33, CDC37, CDC 42, CDC43, CDC53, CDC6, CHC1, CIN8, CKA1, CKA2, CLA4, CLB1, CLB2, CLB3, CLB4, CLB5, CLN1, CLN2, CLN3, CMP2, CNA1, COF1, CTT1, DBF2, DBF20, DPM1, ERG25, GIC1, GIC2, GPA1, GRR1, HCA4, HIS4, HOC1, HSF1, KAR1, KES1, KRE6, KSS1, MBP1, NMT1, ORC2, ORC5, PDE2, PEP12, PEP7, PKC1, P LC1, PMR1, POL30, PRP18, RAM1, RAS1, RAS2, RBL2, RED1, RFC1, RH01, RH03, RH04, SAC1, SEC13, SEC14, SEC22, SEC4, SET1, SIS2, SKP1, SPC98, SRA1, SR04, SRP1, SSA1, SSA2, SSA4, SSN8, STE20, STN 1, STT4, SUJ 3, SWE1, SW14, SW16, TEL1, TOR1, TUB1, TUB4, VMA1, YCK1, YCK2, YPT1.

CELL DAMAGE APN1, BUB1, CDC28, CDC45, CDC46, CDC47, CDC54, CDC7, CLB1, CLB2, CLB3, CLB5, DDC1, DDR2, DDR48, DIN7, DUN1, ECM32, HSM3, IMP2, MEC1, MEC3, MGT1, MOL1, MRE11, MUS81, NTG1, PDS1, PGD1, P HR1, POL2, POL3, POL30, POL4, PR11, PS02, RAD14, RAD16, RAD17, RAD18, RA D24, RAD30, RAD51, RAD52, RAD54, RAD55, RAD57, RAD7, RAD9, RDH54, REV3, RFA1, RFC5, RNR1, RNR2, RNR3, RNR4, RPH1, SIC1, SML1, SP K1, STN1, STS1, TEL1, TFA1, TFA2, TUP1, UBC7, UB14, XBP1, YBR098W, YFH1.

OTHER RELEVANT MUTANTS AND GENES Y-1,9520b, C658-K7, JPD 4, JPM 9, Cy32, E354, JC488, PSY 142, 01-2, Y217, JC787-9A, ML1-21, Y500,86-9C, GL1, GT5-1A, HD565A, PZ1, 127-4D, Y229, JC302-26B, JC482, LB2211-2B, MH41-7B/P21, erg 81, SEY6211, GL4, K335, MK20, MK34, DE4-3A, DE4-3B, DE4-3C, MMY011, UH 1-GRGZ, 2150-2-3a, Y211, DP1/517,943,1117, C658, 1252, H79.20.3, LB1-3B, C658-K42, R29B, LB54-3A, XW520-9A, ade7, D225-5A, 309, SDH1, SDH2, SDH3, SDH4, TCM62, PDE1, PDE2.

Mammalian Genes:

11-beta hydroxysteroid dehydrogenase type II, 12-lipoxygenase, 17-beta hydroxysteroid dehydrogenase, 60S ribosomal protein L6,6-Omethylguanine-DNA methyltransferase, Activating transcription factor 2, Activating transcription factor 3, Activating transcription factor 4, Activin beta E, Activin receptor type 11, Acyl-CoA dehydrogenase, Acyl CoA Carrier Protein, Adenine nucleotide translocator 1, Alanine aminotransferase, Alcohol dehydrogenase 1, Alcohol dehydrogenase 2, Alcohol dehydrogenase 3, Alcohol dehydrogenase 4, Alcohol dehydrogenase 5, Aldehyde dehydrogenase 1, Aldehyde dehydrogenase 2, Aldehyde dehydrogenase 3, Alpha 1-antitrypsin, Alpha-1 acid glycoprotein, Alpha-1 antichymotrypsin, Alpha-catenin, Alphatubulin, Apolipoprotein A1, Apolipoprotein A11, Apolipoprotein Clil, Apolipoprotein E, Aryl hydrocarbon receptor, Aspartate aminotransferase, mitochondrial, Ataxia telangeictasia, ATP-dependent helicase 11 (70 kDa), ATP-dependent helicase 11 (Ku80), BAG-1, BAK, Bax (alpha), Bcl-2, Bcl-xL, Beta-actin, Bilirubin UDP-glucuronosyl-transferase isozyme 1, Bilirubin UDP-glucuronosyl-transferase isozyme 2, Biliverdin reductase, Branched chain acylCoA oxidase, BRCA1, BR-cadherin, C4bbinding protein, c-abl, Calcineurin B, Calnexin, Calprotectin, Calreticulin, canalicular multispecific organic anion transporter, Carbonic Anhydrase 111, Carnitine palmitoyl-CoA transferase, Caspase 1, Caspase 2 (Nedd2), Caspase 3 (CPP32beta), Caspase 5 (ICE relIII), Caspase 6 (Mch2-alpha), Caspase 7 (Mch3alpha), Caspase 8 (FLICE), Catalase, CatecholOmethyltransferase, CCAAT/enhancer-binding protein alpha, CCAAT/enhancer-binding protein epsilon, Cell division cycle protein 2, Cell division cycle protein 20, Cell division cycle protein 25, Cellular retinoic acid binding protein 1, Cellular retinoic acid binding protein 2, cerb; c-fos, Checkpoint kinase-1, Cholesterol esterase, c-H-ras, cjun, Clusterin, c-myc, Complement component C3, Connexin 30, Connexin32, Connexin-40, Corticosteroid binding globulin, Corticotropin releasing factor, C-reactive protein, Creatine kinase b, Cyclin D1, Cyclin dependent kinase 1, Cyclin dependent kinase 4, Cyclin dependent kinase inhibitor 1A, Cyclin E, Cyclin G, Cyclin-dependent kinase 4 inhibitor (P116), Cyclindependent kinase 4 inhibitor B (P16), Cyclin-dependent kinase inhibitor P27Kip1, Cyclooxygenase 2, Cystic fibrosis transmembrane conductance regulator, Cytochrome P450 11A1, Cytochrome P450 17A, Cytochrome P450 1A1, Cytochrome P450 1A2, Cytochrome P450 1 B1, Cytochrome P450 2A1, Cytochrome P450 2A3, Cytochrome P450 2A6, Cytochrome P450 2131, Cytochrome P450 21310, Cytochrome P450 2132, Cytochrome P450 2C11, Cytochrome P450 2C12, Cytochrome P450 2C19, Cytochrome P450 2C9, Cytochrome P450 2D6, Cytochrome P450 2E1, Cytochrome P450 2F2, Cytochrome P450 3A1, Cytochrome P450 3A4, Cytochrome P450 4A, Cytochrome P450 4A1, Damage specific DNA binding protein p48 subunit, Defender against cell death-1, Deleted in colorectal cancer, Deltalike protein, Dihydrofolate reductase, Disulfide isomerase related protein (ERp72), DNA binding protein inhibitor ID2, DNA dependent helicase, DNA dependent protein kinase, DNA ligase 1, DNA ligase IV, DNA mismatch repair protein (MLH1), DNA mismatch repair protein (PMS2), DNA mismatch repair/binding protein (MSH3), DNA polymerase alpha, DNA polymerase beta, DNA polymerase beta, DNA repair and recombination homologue (RAD 52), DNA repair helicase II ERCC-3, DNA repair protein (RAD 50), DNA repair protein (XRCC1), DNA repair protein XP-D, DNA replication factor C (36 kDa), DNA topoisomerase 1, DNA topoisomerase 11, Dopamine beta-hydroxylase, DRA, Dynein light chain 1, E2F, Early growth regulated protein 1, E-Cadherin, ECE-1 (endothelin converting enzyme), Endothelin-1, Enolase alpha, Enoyl CoA hydratase, Eotaxin, Epidermal growth factor, Epoxide hydrolase, ERA-B, ERCC 1 (excision repair protein), ERCC 3 (DNA repair helicase 11), ERCC 5 (excision repair protein), ERCC 6 (excision repair protein), ERK1, Erythropoietin, Erythropoietin receptor, ESelectin, Estrogen receptor, Farnesol receptor, Fas antigen, Fas associated death domain (FADD), Fas ligand, Fas/Apol receptor, Fatty acid synthase, Fatty acyl-CoA oxidase, Fatty acyl-CoA synthase, FEN-1 (endonuclease), Fibrinogen gamma chain, Fibronectin receptor, FIC1, Filagrin, Flavin containing monooxygenase 1, Flavin containing monooxygenase 3, FosB, Fra-1, Fucosyl transferase (alpha-1,2fucosyltransferase), Gadd153, Gadd45, Gamma-glutamyl hydrolase precursor, Gamma-glutamyl transpeptidase, GCLR, GCLS, Glucocorticoid receptor, Glucose-6-phosphate dehydrogenase, Glucose-regulated protein 170, Glucose-regulated protein 58, Glucose-regulated protein 78, Glucoseregulated protein 94, Glutamicoxaloacetic transaminase, Glutaminc-pyruvic transaminase, Glutathione peroxidase, Glutathione reductase, Glutathione S-transferase alpha subunit, Glutathione S-transferase 4a, Glutathione synthetase, Glyceraldehyde 3-phosphate dehydrogenase, GOS24 (zinc finger transcriptional regulator), Granulocyte-macrophage colony-stimulating factor, Growth-arrested-specific protein 1, Growth-arrested-specific protein 3, GT mismatch binding protein, H-cadherin, Heat shock protein 12, Heat shock protein 47, Heat shock protein 70, Heat shock protein 70.1, Heat shock protein 90, Helicase-like transcription factor, Heme binding protein 23, Heme oxygenase-1, Hepatic lipase, Hepatocyte growth factor, Hepatocyte growth factor activator, Hepatocyte growth factor receptor, Hepatocyte nuclear factor 4, Histone 2A, Histone 28, HMG CoA reductase, Hydroxyacyl CoA dehydrogenase, Hydroxysteroid sulfotransferase a, Hypoxanthine-guanine phosphoribosyltransferase, ICE-rel 11 (Caspase 4), ICH-2 cysteine protease=CASPASE 4, IkB-a, Insulin-like growth factor binding protein 1, Insulin-like growth factor binding protein 2, Insulin-like growth factor binding protien 3, Insulin-like growth factor I, Insulin-like growth factor 11, Integrin alpha, Integrin alpha L, Integrin betas, Integrin beta2, Intercellular adhesion molecule-1, Intercellular adhesion molecule-2, Intercellular adhesion molecule-3, Interferon gamma, Interferon inducible protein 10, Interferon inducible protein 15, Interleukin-1 alpha, Interleukin-12, Interleukin-2, Interleukin-4, Interleukin-5, Interleukin-6, Involucrin, JNK1 stress activated protein kinase, K-cadherin, Ki67, Lactate Dehydrogenase 8, Lactoferrin, Lipopolysaccharide binding protein, Lipoprotein lipase precursor, Liver fatty acid binding protein, L-myc, Low density lipoprotein receptor, Luteinizing hormone, Lysyl oxidase, Macrophage inflammatory protein-1 alpha, Macrophage inflammatory protein-1 beta, Macrophage inflammatory protein-2 alpha, Macrophage inflammatory protein-2 beta, Macrophage inflammatory protein-3 alpha, Macrophage inflammatory protein-3 beta, Malic enzyme, MAP kinase kinase, Matrix metal loproteinasel, Matrix metal loproteinase-2, MDM-2, MET proto-oncogene, Metallothionein 1, Metallothionein 2, Metallothionein 3, Metallothionein IA, Metallothionein IG, Metalregulatory transcription factor-1, Mitogen activated protein kinase (P38), Mitogen inducible gene (mig-2), MOAT-B (MRP/organic anion transporter), Monoamine oxidase A, Monoamine oxidase B, Multidrug resistance-associated protein, Multidrug resistant protein-1, Multidrug resistant protein-2, Multidrug resistant protein-3=cMOAT2, MUTL homologue (MLH1), MutS Homologue (MSH2), Myeloid cell differentiation protein-1, Na/taurocholate cotransporting polypeptide, NADPH cytochrome P450-oxidoreductase, NADPH cytochrome P450 reductase, NADPH quinone oxidoreductase-1 (DTDiaphorase), Natural killer cell-enhancing factor B, N-cadherin, NF-kappaB (p65), Nitric oxide synthase-1, inducible, Nucleoside diphosphate kinase beta isoform, 0-6-alkylguanine-DNAalkyltran-sferase, OBcadherin 1, OB-cadherin 2, Octamer binding protein 1, Octamer binding protein 2, Octamer binding protein 3, Oncostatin M, Organic anion transporter 1, Organic anion transporter 3, Organic anion transporter K1, Organic anion transporting polypeptide 1, Organic cation transporter 1, Organic cation transporter 2, Organic cation transporter 3, Organic cation transporter N1, Organic cation transporter N2, Ornithine decarboxylase, Osteopontin, Oxygen regulated protein 150, p53, PAPS synthetase, P-cadherin, PEGS (progression elevated gene 3), Peroxisomal 3-ketoacyl-CoA thiolase 1, Peroxisomal 3-ketoacylCoA thiolase 2, Peroxisomal acyl-CoA oxidase, Peroxisomal fatty acyl-CoA oxidase, Peroxisome assembly factor 1, Peroxisome assembly factor 2, Peroxisome biogenesis disorder protein-1, Peroxisome biogenesis disorder protein-11, Peroxisome biogenesis disorder protein-4, Peroxisome hydratase, Peroxisome proliferator activated receptor alpha, Peroxisome proliferator activated receptor gamma, Phenol sulfotransferase, Phosphoenolpyruvate carboxykinase, Phosphoglyceride kinase, Phospholipase A2, Plasminogen activator inhibitor 2, Platelet derived growth factor B, Platelet/endothelial cell adhesion molecule-1, Poly (ADP ribose) polymerase, Proliferating cell nuclear antigen gene, Prostaglandin H synthase, Protein kinase C betal, Protein-tyrosine phosphatase, Putative protein tyrosine phosphatase, RAID, RAID 51 homologue, RANTES, Ref 1, Replication factor C, 40-kDa subunit (Al), Replication protein A (70 kDa subunit), Retinoblastoma, Retinoblastoma related protein (P 107), Retinoid X receptor alpha, Retinoid X receptor beta, Retinoid X receptor gamma, Ribonucleotide reductase M1 subunit, Ribosomal protein L13A, Ribosomal protein S9, RNA-dependent helicase, ROAT1 (renal organic anion transporter), Serum amyloid A1, Serum amyloid A2alpha, Sister of p-glycoprotein, Sodium/bile acid cotransporter, Sonic hedgehog gene, SQM1, Superoxide Dismutase Cu/Zn, Superoxide dismutase Mn, T-cell cyclophilin, Tenascin, Thiopurine methyltransferase, Thioredoxin, Thrombospondin 2, Thymidine kinase, Thymidylate synthase, Thymosin beta-10, Tissue inhibitor of metalloproteinases-1, Tissue transglutaminase, Transcription factor IID, Transferrin, Transforming growth factor-beta 3, Tumor necrosis factor associated factor 2 (TRAF2), Tumor necrosis factor receptor 1, Tumor necrosis factor receptor 2, Tumor necrosis factor receptor-1 associated protein (TRADD), Tumor necrosis factor-alpha, Tumor necrosis factor beta, Type 1 interstitial collagenase, Tyrosine aminotransferase, Tyrosine protein kinase receptor (UFO), Ubiquitin, Ubiquitin conjugating enzyme (Rad 6 homologue), Ubiquitin-homology domain protein PIC1, UDPglucuronosyltransferase 1, UDP-glucuronosyltransferase 1A6, UDPglucuronosyltransferase 2, UDP-glucuronosyltransferase 28, Uncoupling protein 1, Uncoupling protein 2, Uncoupling protein 3, Urate oxidase, UV excision repair protein RAD 23 (XP-C), Vascular cell adhesion molecule 1 (VCAM-1), Vascular endothelial growth factor, Vascular endothelial growth factor D, Very long-chain acyl-CoA dehydrogenase, Vimentin, Vitellogenin, Wafl, XRCC1 (DNA repair protein).

Passenger genes also include genes encoding prodrug-activating enzymes such as viral thymidine kinase, viral thymidine phosphorylase, cytosine deaminase, bacterial carboxypeptidase G2, cytochrome P450 proteins, carboxylesterase, deoxycytidine kinase, nitroreductase, purine nucleoside phosphorylase, horseradish peroxidase, and xantine guanine phosphoribosyl transferase. Also included are genes encoding “proteotoxins” such as diphtheria toxin and cytolethal distending toxin.

The present invention, thus generally described, may be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES Example 1 Construction of a Conditionally Replicating Adenovirus Pair of the Invention Using a Heat-Activated, Mifepristone-Dependent Gene Switch

Adenovirus is abbreviated Ad below. Generally known molecular biology and biochemistry methods are used. Molecular biology methods are described, e.g., in “Current protocols in molecular biology, Ausubel et al., eds., volumes 1-4, John Wiley and Sons, Inc. ISBN 0-471-50338-X.

Recombinant Adenovirus 1 (rAd1)

Recombinant adenovirus 1 lacks nucleotides 28,130-30,820 encompassing E3. Nucleotide numbers relating to the adenovirus type 5 genome are as defined in GI: 33694637. Davison et al. 2003. J. Gen. Virol. 84, 2895-2908. Further, E1A promoter and E4 promoter sequences are replaced with a GAL4 site-containing minimal promoter. According to the convention followed elsewhere in this specification, rAd1 corresponds to a second virus of a conditionally replicating virus pair of the invention.

The simplified system for generating recombinant adenovirus developed by He et al. (Proc. Natl. Acad. Sci. USA 95, 2509-2514 (1998)) was employed to construct rAd1 (rAd2). This system was made available commercially by Stratagene Corp. of La Jolla, Calif. A manual entitled “AdEasy™ Adenoviral Vector System” (revision no. 060002) is distributed by the Company to its customers and is also available at the Company's website.

Mutagenesis was carried out in transfer vector pShuttle (He et al. 1998. Proc. Natl. Acad. Sci. USA 95, 2509-2514) distributed by Statagene Corporation. FIG. 7 presents the complete nucleotide sequence of this plasmid. According to Stratagene's manual entitled “AdEasy™ Adenoviral Vector System” (revision no. 060002), pShuttle contains the following adenovirus sequence elements: left inverted terminal repeat, encapsidation signal (Ad 1-331), “right arm homology region” (3,534-5790), “left arm homology region” (34,931-35,935) and right inverted terminal repeat. A site-directed mutagenesis procedure, the so-called QuikChange® procedure of Stratagene Corp. that makes use of the polymerase chain reaction (PCR) was employed to delete E4 promoter sequences present in the left arm homology region of pShuttle (Ad 35,586-35,808) and replace them by an NheI restriction site. The QuikChange protocol is described, for example, in instruction manual “QuikChange XL site-directed mutagenesis kit” (revision no. 063003) published by Stratagene Corp. at their website and distributed to customers by mail. A related procedure is described briefly in Guettouche, T. (2002). Ph.D. Thesis, University of Miami, Miami, Fla. The resulting construct was labeled “pShuttle-Nhe”.

A GAL4-binding site-containing minimal promoter was inserted into the NheI site of pShuttle-Nhe. To achieve this, a DNA fragment encompassing nucleotides 7677 to 323 of plasmid pGene/V5-His (Invitrogen Corporation of Carlsbad, Calif.) not including a complete PacI site was PCR-amplified using appropriate primers also containing an NheI restriction site. After digestion of the PCR fragment with NheI, it was ligated to NheI-digested pShuttle-Nhe DNA. Recombinants were identified by restriction digestion and nucleotide sequencing. Based on restriction analysis and nucleotide sequencing results, a clone was selected in which the inserted promoter sequence was correctly oriented relative to the E4 sequences in the left arm homology region of pShuttle-Nhe. This plasmid was named pShuttle-Nhe-GAL. The nucleotide sequence of pGene/V5-His is provided in FIG. 8. The plasmid is also described in instruction manual “GeneSwitch™ System, Version B, 000829/25-0313” of Invitrogen Corp. that is distributed by the Company to its customers and is also available at the Company's website.

Next, the right arm homology region of pShuttle-Nhe-GAL was replaced with Ad sequences also containing the beginning of the E1 region in addition to the homology region. First, pShuttle-Nhe-GAL was digested with BglII and PmeI, filled in with DNA polymerase Klenow fragment and religated to produce pShuttle-Nhe-GAL-delE1. The latter plasmid lacked the right arm homology region. Next, a fragment containing Ad 496-5780 was obtained by PCR amplification from plasmid pXC1 (Microbix Corporation, Toronto, ON). The nucleotide sequence of this plasmid is presented as FIG. 9. The latter PCR amplification was carried out using a forward primer (reading into the E1 gene) containing AscII and NotI restriction sites and a reverse primer containing a NotI restriction site. After digestion with NotI, the PCR fragment was ligated to NotI-digested pShuttle-Nhe-GAL-delE1 DNA to produce plasmid pShuttle-Nhe-GAL-E1. A clone was chosen in which the orientation of the inserted E1 sequence was the same as that of the substituted truncated E1 sequence. In a subsequent subcloning step, a DNA fragment encompassing nucleotides 7677 to 323 of plasmid pGeneN5-His was PCR-amplified using appropriate primers also containing an AscII restriction site. After digestion of the PCR fragment with AscII, it was ligated to AscII-digested pShuttle-Nhe-GAL-E1 DNA. Recombinants were identified by restriction digestion and by nucleotide sequencing. Based on restriction analysis and nucleotide sequencing information, a clone was selected that contained the inserted pGene/V5-His promoter sequence in the correct orientation relative to the E1 sequences. This clone was named pShuttle-Nhe-GAL-E1-GAL. Finally, the NotI site next to the inserted GAL4 site-containing promoter (inserted pGene/V5-His promoter sequence) was deleted by QuikChange mutagenesis. As a consequence, the resulting construct pShuttle-Nhe-GAL-E1-GAL-delNot contained a unique NotI site situated immediately downstream from the E1 sequences. To prepare recombinant Ad, pShuttle-Nhe-GAL-E1-GAL-delNot DNA was linearized by NotI digestion and was co-electroporated with pAdEasy-1 DNA (He et al. 1998. Proc. Natl. Acad. Sci. USA 95, 2509-2514) into E.coli BJ5183 cells. BJ5183 cells (Stratagene catalog no. 200154) have the cellular components necessary to carry out homologous recombination between introduced viral sequences. Detailed methods for the generation of recombinant Ad plasmids and the subsequent production of recombinant Ad viruses are discussed in He et al. (Proc. Natl. Acad. Sci. USA 95, 2509-2514 (1998)) and in Stratagene manual“AdEasy™ Adenoviral Vector System” (revision no. 060002). Briefly, recombinant Ad plasmids were characterized by restriction digestion. After preparation of a sufficient amount of DNA of a correct recombinant, the DNA was digested with PacI to separate plasmid sequences and inserted sequences comprising the Ad sequences. To produce recombinant Ad, the digested DNA was transfected into 293E4pIX cells. 293E4pIX cells are 293 cells containing an MMTV promoter-driven E4 transcription unit. In the presence of dexamethasone, these cells provide both E1 and E4 functions that were needed to amplify the desired recombinant virus that has conditionally active E1A and E4 genes. Using standard technology (briefly described in He et al.; see also Graham and Prevec. 1991. Manipulation of adenovirus vectors. In: Methods in Molecular Biology, Gene Transfer and Expression Protocols, vol. 7, ed. Murray, The Humana Press Inc., Clifton, N.J., pp. 109-127), virus plaques were isolated, and rAd1 stocks were prepared. Viral stocks can be purified by CsCl gradient centrifugation or chromatographic procedures.

Note that pAdEasy-1 contains all Ad sequences except for nucleotides 1-3,533 and 28,130-30,820. Hence, rAd1 (and rAd2, described below) contains a deletion of a nonessential E3 region. By using an Ad plasmid that contains all E3 sequences in the recombination step, a recombinant Ad1 version that has a complete E3 region could be obtained.

Recombinant Adenovirus 2 (rAd2)

Recombinant adenovirus 2 is an E1/E3/E4-deleted Ad (lacking Ad 1-3,533, 28,130-30,820 and 35,586-35,808) that contains a transactivator that is closely related to transactivator GLVP described before. The transactivator employed in this example is a GAL4 DNA-binding domain-modified human progesterone receptor ligand-binding domain-human p65 activation domain chimera (GLP65) that was retrieved from plasmid pSwitch (Invitrogen Corp.). The complete sequence of pSwitch is shown in FIG. 10. The transactivator is activated by estrogen antagonist mifepristone. In rAd2, the transactivator gene is functionally linked to an hsp70B-GAL4 tandem promoter. Optionally, rAd2 may also contain a passenger gene that is regulated by the hsp70B-GAL4 tandem promoter or another promoter. According to the convention followed elsewhere in this specification, rAd2 corresponds to a first virus of a conditionally replicating virus pair of the invention.

Construction of an hsp70-GAL4 tandem promoter. Plasmid hsp70-fLuc was obtained by subcloning promoter and RNA leader sequences of the human Hsp70B gene from OR173 into a plasmid containing a firefly luciferase gene. Voellmy et al. 1985. Proc. Natl. Acad. Sci. USA 82, 4949-4953. Hsp70-fLuc contains more than 2 kbp of hsp70B gene nontranscribed sequence. A short fragment of about 460 bp can be removed from the latter sequence using restriction enzymes BamHI and HindIII that will contain the essential promoter sequence (about 350 bp) that includes the three functionally important heat shock element sequences, the Hsp70B transcription start site and an about 110 bp-long transcribed sequence (RNA leader region). Voellmy et al. 1985. Proc. Natl. Acad. Sci. USA 82, 4949-4953. Schiller et al. 1988. J. Mol. Biol. 203, 97-105.

To prepare hsp70-GAL4-fLuc, an SgfI site was inserted in the hsp70B 5′ untranslated region present in hsp70-fLuc by the QuikChange procedure (Stratagene) using primer 5′-CAGCCTCCGTGGCCTCGCGATCGCAGCATCCGACAAGAAGC (SEQ ID NO: 5) and its complement (hsp70-GAL4-fLuc/Sgf). A short intron sequence (about 245 bp) was PCR-amplified from construct RL-CMV (Promega) using primers 5′-TATGCGATCGCTTCTGACACAACAGTCTCG (SEQ ID NO: 6) and 5′-TATGCGATCGCCTTAAGAGCTGTAATTGAAC (SEQ ID NO: 7), digested with SgfI and ligated to SgfI-digested hsp70-GAL4-fLuc/Sgf. Resulting construct hsp70-GAL4-fLuc/Sgf/Int served as template in a QuikChange reaction designed to introduce an AscI restriction site into the intron sequence (hsp70-GAL4-fLuc/Sgf/Int/Asc). Primers employed were 5′-TATGGCGCGCCACCTGACGTCGACGG (SEQ ID NO: 8) and 5′-ATAGGCGCGCCGGTAAGCTTAAGTTTAAACGCTAGC (SEQ ID NO: 9). Finally, a sequence (about 450 bp) containing a minimal GAL4 promoter was PCR-amplified from construct GeneV5-HisA (Invitrogen) using primers 5′-TATGGCGCGCCTCGACGGATCGGGAGATC (SEQ ID NO: 10) and 5′-TATGGCGCGCCTGTTAATTAACACGGGG (SEQ ID NO: 11), digested with AscI and ligated to AscI-cut hsp70-GAL4-fLuc/Sgf/Int/Asc DNA, yielding hsp70-GAL4-fLuc.

For the construction of hsp70-GAL4-GLP65, a QuikChange reaction using primer 5′-CGAGTCTAGAGATATCGAATTCAGGCGCGCCTTGTCGACTCGAAGATCTG (SEQ ID NO: 12) and its complement was performed to insert an AscI site downstream of the multicloning site of construct Shuttle (Stratagene). The resulting construct was named AI. A glp65 gene fragment was PCR-amplified from construct Switch (Invitrogen) using primers 5′-TATTCTAGAACCAAGCTACCGGTCCACC (SEQ ID NO: 13) and 5′-ATAGGCGCGCCTCAGAAGCCATAGAGCCC (SEQ ID NO: 14), digested with AscI and XbaI, and ligated to AscI/XbaI-cut A1 DNA to produce construct 1A. Finally, an hsp70-GAL4 promoter cassette was excised from hsp70-GAL-fLuc using BamHI and HindIII. The filled promoter cassette fragment was ligated to XbaI-digested and filled construct 1A DNA. To construct GAL4-fLuc, a HindIll(filled)-XhoI fragment from 17×4 TATA CAT including a GAL4-responsive promoter was inserted in between the SmaI and XhoI sites of GL2B (Promega Corp.). For a description of construct 17×4 TATA CAT see Wang et al. 1997. Nat. Biotechnol. 15, 239-243; Wang et al. 1997. Gene Therapy 4, 432-441.

The gene switch consisting of hsp70-GAL4-GLP65 and the GLP65-responsive GAL4 promoter of construct GAL4-fLuc was tested extensively in transient transfection experiments, stable cell lines containing the gene switch as well as animal experiments in which hsp70-GAL4-GLP65 and GAL4-fLuc were introduced in gastrocnemius muscles of mice by injection and in vivo electroporation. Collectively, these experiments demonstrated that the activity of target gene fLuc was stringently regulated by the gene switch in vitro and in vivo. fLuc expression was strongly induced in cells that were heat-treated and exposed to mifepristone. Essentially no activity was measured in cells that were left untreated, were heat-treated only, or were only exposed to mifepristone in the absence of a heat treatment. Induced activity remained high for at least one weak after a single, transient activating heat treatment in the continued presence of mifepristone. Mifepristone removal resulted in deactivation of the gene switch.

Several derivatives of hsp70-GAL4-GLP65 constructs were also prepared and used for construction of different versions of rAd2. A preferred construct that displays a particularly low background level of transactivation ability in the absence of heat activation and mifepristone is Hsp70/GAL4-GLP65-RKC. In this construct, the Kozak consensus sequence of the glp65 gene was mutated to reduce translation initiation efficiency, and nontranslated sequences 3′ of the GLP65-coding sequence were removed. Intermediary construct hsp70-GAL4-GLP65-34C was prepared by QuikChange mutagenesis of Hsp70-GAL4-GLP65, using oligonucleotide 5′-ACCAAGCTACCGGTCCTCCATGTACTCCCAGCAGCCAGATCTGAAGC-3′ (SEQ ID NO: 15) and its complement as primers. For obtaining hsp70-GAL4-GLP65-RKC, the hsp70-GAL4-GLP65-34C gene was PCR-amplified using primers 5′-ATACCTAGGCGGATCCGACCTCCCACAGC (SEQ ID NO: 16) and 5′-ATAGCGGCCGCCCGGAGGATCCTTAGGAGC (SEQ ID NO: 17), digested with AvrII and NotI, and ligated to NotI/XbaI-digested vector Shuttle. All subcloning and mutagenesis steps were monitored by restriction analysis and nucleotide sequencing.

To prepare the actual shuttle constructs used in the construction of different versions of rAd2, the hsp70B-GAL4-GLP65 gene cassette and derivative genes including hsp70-GAL4-GLP65-RKC were PCR-amplified and inserted into the multicloning site of pShuttle-Nhe (described under rAd1). The counterclockwise orientation of the genes in the resulting constructs was verified by restriction analysis and nucleotide sequencing. Constructs were named SNPB3DE4xGAL-3 (hsp70B-GAL4-GLP65), or SNKC6DE4xGAL-7 (hsp70-GAL4-GLP65-RKC). The same designations are also used to identify particular rAd2s.

It is noted that the above shuttle constructs may also include a passenger gene such as, for example, an i112 gene. IL12-coding sequences and 3′ untranslated sequences could be PCR-amplified from an appropriate construct. This PCR fragment and a fragment containing the hsp70B-GAL4 tandem promoter could be inserted in the multicloning site next to the transactivator cassette. Nucleotide sequence analysis could be used to verify that the hsp70-GAL4 promoter is properly situated to control transcription from the i112 gene. Alternatively, a single tandem promoter may be used to control glp65 and i112 gene activity. In this case the sequence of elements inserted in pShuttle-Nhe could be as follows: hsp70B-GAL4 tandem promoter—i112 gene—internal ribosome binding site—glp65 gene—3′ untranslated sequences. Attention should be paid to the positioning of a passenger gene that is regulated by a constitutively active promoter. In such a case, a passenger gene cassette should be inserted downstream from the glp65 gene or in an orientation that results in an opposite transcriptional direction from that of the glp65 gene. It is noted further that any passenger gene and associated 3′ untranslated sequences to be incorporated in an rAd2 need to be screened for the presence of PmeI and PacI sites. If such sites are present, they need to be deleted. Deletion can typically be achieved conveniently by using the QuikChange mutagenesis procedure.

To prepare rAd2 (virus), constructs SNPB3DE4xGAL-3 and SNKC6DE4xGAL-7 were linearized with PmeI and co-introduced with AdEasy-1 sequences in an appropriate E.coli strain to achieve homologous recombination of viral sequences. Isolation of correct recombinant plasmids, preparation of sufficient amounts of plasmids, digestion with PacI, transfection into 293 cells expressing E4, and isolation of rAd2 viruses were as described before for rAd1.

Example 2 Demonstration of Conditional Replication of a rAd1/rAd2 Virus Pair of the Invention

Parallel, subconfluent, adherent cultures of human HeLa cells grown under standard conditions were infected with a rAd1/rAd2 (SNPB3DE4xGAL-3) combination at a total multiplicity of infection of about 0.1. rAd1/rAd2 ratio in this particular experiment was 5:1. Medium was changed one day after infection, and cultures were either heat-treated at 42° C. for 2 h or not treated and then cultured for 8 days in the presence or absence of 10 nM mifepristone. Cells were diluted (split) once about halfway through the incubation period. At the end of the 8-day incubation, cells were fixed and stained using anti-hexon protein antibody. Results revealed readily detectable spreading of virus through cultures that had been heat-treated and postincubated in the presence of mifepristone but not through cultures that had not been heat-treated or had not received mifepristone.

Example 3 Demonstration of Oncolytic Action of a rAd1/rAd2 Virus Pair of the Invention

Parallel, subconfluent, adherent cultures of human HeLa cells grown under standard conditions were infected with a rAd1/rAd2 (SNPB3DE4xGAL-3) combination at a total multiplicity of infection of about 25. rAd1/rAd2 ratio in this particular experiment was 1:1. Cultures were either heat-treated at 43° C. for 2 h or not treated and then cultured for 4 days in the presence or absence of 10 nM mifepristone. At the end of this period, cells were stained with crystal violet, and cell numbers were estimated. Quantitative killing of cells was observed only in cultures that had been heat-treated and exposed to mifepristone.

All references cited in this application, including publications, patents and patent applications, shall be considered as having been incorporated in their entirety. 

1. A conditionally replicating pair of modified viruses consisting of (a) a first virus that is defective in one or more genes required for efficient replication and whose genome includes a gene for a small-molecule-regulated transactivator, the gene for the small-molecule-regulated transactivator being functionally linked to a nucleic acid sequence that acts as a heat shock promoter, and (b) a second virus comprising all genes required for efficient replication of which genes at least one gene is under the control of a promoter that is responsive to the small molecule-regulated transactivator of the first virus and is not complemented by a functional gene of the first virus, wherein expression of all genes of the second virus required for its replication will complement the defects of the first virus and result in its replication.
 2. The pair of modified viruses of claim 1, wherein the nucleic acid sequence functionally linked to the gene for the small-molecule-regulated transactivator also acts as a promoter that is responsive to the transactivator.
 3. The pair of modified viruses of claim 1, wherein the genome of the first virus further includes a passenger gene.
 4. The pair of modified viruses of claim 1, wherein both viruses are members of the same family selected from the group consisting of Adenoviridae, Herpesviridae, and Retroviridae.
 5. The pair of modified viruses of claim 1, wherein the first virus is derived from an adeno-associated virus and the second virus from an adenovirus.
 6. The modified virus pair according to claim 5, wherein the small-molecule-regulated transactivator controls the expression by the second virus of at least one protein selected from the group consisting of E1A, E1B and E4.
 7. The pair of modified viruses of claim 1, wherein both viruses are adenoviruses.
 8. The modified virus pair according to claim 7, wherein the small-molecule-regulated transactivator controls the expression by the second virus of at least one protein selected from the group consisting of E1A, E1B and E4.
 9. The modified virus pair according to claim 1, wherein at least one gene required for efficient replication is not present or is nonfunctional in the second virus but is present and functional in the first virus. 